Dimeric IAP inhibitors

ABSTRACT

Molecular mimics of Smac are capable of modulating apoptosis through their interaction with cellular IAPs (inhibitor of apoptosis proteins). The mimetics are based on a monomer or dimer of the N-terminal tetrapeptide of IAP-binding proteins, such as Smac/DIABLO, Hid, Grim and Reaper, which interact with a specific surface groove of IAP. Also disclosed are methods of using these peptidomimetics for therapeutic purposes. In various embodiments of the invention the Smac mimetics of the invention are combined with chemotherapeutic agents, including, but not limited to topoisomerase inhibitors, kinase inhibitors, NSAIDs, taxanes and platinum containing compounds use broader language.

CROSS REFERENCES AND RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/196,202 entitled DIMERIC IAP INHIBITORS filed Aug. 2, 2011, whichis a continuation application of U.S. application Ser. No. 12/403,915(now U.S. Pat. No. 8,022,230) entitled DIMERIC IAP INHIBITORS filed onMar. 13, 2009, which is a continuation application of U.S. applicationSer. No. 11/363,387 (now U.S. Pat. No. 7,517,906) entitled DIMERIC IAPINHIBITORS filed on Feb. 27, 2006, which claims priority from U.S.Provisional Application No. 60/656,201 entitled “PEPTIDOMIMETICS” filedon Feb. 25, 2005, U.S. Provisional Application No. 60/668,344 entitled“IMMUNOTHERAPEUTIC USES OF SMAC MIMETICS” filed on Apr. 5, 2005, U.S.Provisional Application No. 60/692,111 entitled “PEPTIDOMIMETICS OF SMACALONE OR IN COMBINATION WITH TOPOISOMERASE INHIBITORS” filed on Jun. 20,2005, U.S. Provisional Application No. 60/706,649 entitled“PEPTIDOMIMETICS OF SMAC AS CAP INHIBITORS” filed on Aug. 9, 2005 andU.S. Provisional Application No. 60/729,853 entitled “PEPTIDOMIMETICS OFSMAC ALONE OR IN COMBINATION WITH PLATINUM CONTAINING COMPOUNDS ANDTAXANES” filed on Oct. 25, 2005 all of which are herein incorporated byreference in their entireties.

BACKGROUND

Apoptosis (programmed cell death) plays a central role in thedevelopment and homeostasis of all multi-cellular organisms. Apoptotiscan be initiated within a cell from an external factor such as achemokine (an extrinsic pathway) or via an intracellular event such asDNA damage (an intrinsic pathway). Alterations in apoptotic pathwayshave been implicated in many types of human pathologies, includingdevelopmental disorders, cancer, autoimmune diseases, as well asneuro-degenerative disorders. One mode of action of chemotherapeuticdrugs is cell death via apoptosis.

Apoptosis is conserved across species and executed primarily byactivated caspases, a family of cysteine proteases with aspartatespecificity in their substrates. These cysteine containing aspartatespecific proteases) (“caspases”) are produced in cells as catalyticallyinactive zymogens and are proteolytically processed to become activeproteases during apoptosis. Once activated, effector caspases areresponsible for proteolytic cleavage of a broad spectrum of cellulartargets that ultimately lead to cell death. In normal surviving cellsthat have not received an apoptotic stimulus, most caspases remaininactive. If caspases are aberrantly activated, their proteolyticactivity can be inhibited by a family of evolutionarily conservedproteins called IAPs (inhibitors of apoptosis proteins).

The TAP family of proteins suppresses apoptosis by preventing theactivation of procaspases and inhibiting the enzymatic activity ofmature caspases. Several distinct mammalian IAPs including XIAP, c-IAP1,c-IAP2, ML-IAP, NAIP (neuronal apoptosis inhibiting protein), Bruce, andsurvivin, have been identified, and they all exhibit anti-apoptoticactivity in cell culture. IAPs were originally discovered in baculovirusby their functional ability to substitute for P35 protein, ananti-apoptotic gene. IAPs have been described in organisms ranging fromDrosophila to human, and are known to be overexpressed in many humancancers. Generally speaking, IAPs comprise one to three Baculovirus LAPIAP repeat (BIR) domains, and most of them also possess acarboxyl-terminal RING finger motif. The BIR domain itself is a zincbinding domain of about 70 residues comprising 4 alpha-helices and 3beta strands, with cysteine and histidine residues that coordinate thezinc ion. It is the BIR domain that is believed to cause theanti-apoptotic effect by inhibiting the caspases and thus inhibitingapoptosis. XIAP is expressed ubiquitously in most adult and fetaltissues. Overexpression of XIAP in tumor cells has been demonstrated toconfer protection against a variety of pro-apoptotic stimuli andpromotes resistance to chemotherapy. Consistent with this, a strongcorrelation between XIAP protein levels and survival has beendemonstrated for patients with acute myelogenous leukemia.Down-regulation of XIAP expression by antisense oligonucleotides hasbeen shown to sensitize tumor cells to death induced by a wide range ofpro-apoptotic agents, both in vitro and in vivo. Smac/DIABLO-derivedpeptides have also been demonstrated to sensitize a number of differenttumor cell lines to apoptosis induced by a variety of pro-apoptoticdrugs.

In normal cells signaled to undergo apoptosis, however, the IAP-mediatedinhibitory effect must be removed, a process at least in part performedby a mitochondrial protein named Smac (second mitochondrial activator ofcaspases). Smac (or, DIABLO), is synthesized as a precursor molecule of239 amino acids; the N-terminal 55 residues serve as the mitochondriatargeting sequence that is removed after import. The mature form of Smaccontains 184 amino acids and behaves as an oligomer in solution. Smacand various fragments thereof have been proposed for use as targets foridentification of therapeutic agents.

Smac is synthesized in the cytoplasm with an N-terminal mitochondrialtargeting sequence that is proteolytically removed during maturation tothe mature polypeptide and is then targeted to the inter-membrane spaceof mitochondria. At the time of apoptosis induction, Smac is releasedfrom mitochondria into the cytosol, together with cytochrome c, where itbinds to IAPs, and enables caspase activation, therein eliminating theinhibitory effect of IAPs on apoptosis. Whereas cytochrome c inducesmultimerization of Apaf-1 to activate procaspase-9 and -3, Smaceliminates the inhibitory effect of multiple IAPs. Smac interacts withessentially all IAPs that have been examined to date including XIAP,c-IAP1, c-IAP2, and ML-IAP. Thus, Smac appears to be a master regulatorof apoptosis in mammals.

It has been shown that Smac acts as an IAP antagonist promoting not onlythe proteolytic activation of procaspases, but also the enzymaticactivity of mature caspase, both of which depend upon its ability tointeract physically with IAPs. X-ray crystallography has shown that thefirst four amino acids (AVPI) of mature Smac bind to a portion of IAPs.This N-terminal sequence is essential for binding IAPs and blockingtheir anti-apoptotic effects.

The basic biology IAP antagonists suggest that they may complement orsynergize other chemotherapeutic/anti-neoplastic agents and/orradiation. Chemotherapeutic/anti-neoplastic agents and radiation wouldbe expected to induce apoptosis as a result of DNA damage and/or thedisruption of cellular metabolism.

Current trends in cancer drug design focus on selective activation ofapoptotic signaling pathways within tumors while sparing normal cells.The tumor specific properties of specific antitumor agents, such asTRAIL have been reported. The tumor necrosis factor-relatedapoptosis-inducing ligand (TRAIL) is one of several members of the tumornecrosis factor (TNF) superfamily that induce apoptosis through theengagement of death receptors. TRAIL interacts with an unusually complexreceptor system, which in humans comprises two death receptors and threedecoy receptors. TRAIL has been used as an anti-cancer agent alone andin combination with other agents including chemotherapeutic drugs andionizing radiation. TRAIL can initiate apoptosis in cells thatoverexpress the survival factors Bcl-2 and Bcl-XL, and may represent atreatment strategy for tumors that have acquired resistance tochemotherapeutic drugs. TRAIL binds its cognate receptors and activatesthe caspase cascade utilizing adapter molecules such as FADD. Currently,five TRAIL receptors have been identified. Two receptors TRAIL-R1 (DR4)and TRAIL-R2 (DR5) mediate apoptotic signaling, and three non-functionalreceptors, DcR1, DcR2, and osteoprotegerin (OPG) may act as decoyreceptors. Agents that increase expression of DR4 and DR5 may exhibitsynergistic anti-tumor activity when combined with TRAIL.

The beneficial effects of TRAIL production have been shown in severaltypes of cancer. For example, intravesical instillation of the BCGvaccine induces a Th1 immune response, resulting in the production ofanti-tumor cytokines, including TRAIL, and the infiltration of thelesion with immune cell and is the first line of therapy for thetreatment of superficial bladder cancer. In vitro studies indicate thatinterferon alpha (INF-α), which in currently being tested in clinicalstudies for efficacy in bladder cancer, causes apoptosis mediated by theautocrine production of TRAIL in human bladder cancer cell lines. Thecirculating level of osteoprotogerin, a decoy receptor for TRAIL, isalso increased in patients with bladder cancer and negatively correlatewith tumor stage, grade and prognosis.

Moreover, it has been shown that TRAIL expression by NK (Natural Killer)cells is enhanced by IL-2 (Interleukin 2) treatment, and the expressionof TRAIL is required for full tumor cell cytotoxic effects. IL-2, acytokine, is currently approved for the treatment of both melanoma andrenal cell carcinoma.

Inhibition of cancer cell replication and/or DNA damage repair willenhance nuclear DNA fragmentation, thus inducing the cell to enter theapoptotic pathway. Topoisomerases, a class of enzymes that reducesupercoiling in DNA by breaking and rejoining one or both strands of theDNA molecule, are vital to cellular processes, such as DNA replicationand repair. Inhibition of this class of enzymes impairs the cellsability to replicate as well as to repair damaged DNA and activates theintrinsic apoptotic pathway.

The main pathways leading from topoisomerase-mediated DNA damage to celldeath involve activation of caspases in the cytoplasm by proapoptoticmolecules released from mitochondria, such as Smac. The engagement ofthese apoptotic effector pathways is tightly controlled by upstreamregulatory pathways that respond to DNA lesions-induced by topoisomeraseinhibitors in cells undergoing apoptosis. Initiation of cellularresponses to DNA lesions-induced by topoisomerase inhibitors is ensuredby protein kinases that bind to DNA breaks. These kinases (non-limitingexamples of which include Akt, JNK and P38) commonly called “DNAsensors” mediate DNA repair, cell cycle arrest and/or apoptosis byphosphorylating a large number of substrates, including severaldownstream kinases.

Platinum chemotherapy drugs belong to a general group of DNA modifyingagents. DNA modifying agents may be any highly reactive chemicalcompound that bonds with various nucleophilic groups in nucleic acidsand proteins and cause mutagenic, carcinogenic, or cytotoxic effects.DNA modifying agents work by different mechanisms, disruption of DNAfunction and cell death; DNA damage/the formation of cross-bridges orbonds between atoms in the DNA; and induction of mispairing of thenucleotides leading to mutations, to achieve the same end result. Threenon-limiting examples of platinum containing DNA modifying agents arecisplatin, carboplatin and oxaliplatin.

Cisplatin is believed to kill cancer cells by binding to DNA andinterfering with its repair mechanism, eventually leading to cell death.Carboplatin and oxaliplatin are cisplatin derivatives that share thesame mechanism of action. Highly reactive platinum complexes are formedintracellularly and inhibit DNA synthesis by covalently binding DNAmolecules to form intrastrand and interstrand DNA crosslinks.

Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to induceapoptosis in colorectal cells. NSAIDS appear to induce apoptosis via therelease of Smac from the mitochondria (PNAS, Nov. 30, 2004, vol.101:16897-16902). Therefore, the use of NSAIDs in combination with Smacmimetics would be expected to increase the activity each drug over theactivity of either drug independently.

U.S. Pat. No. 6,992,063 to Shi et al. entitled “Compositions and methodfor Regulating Apoptosis” filed on Sep. 28, 2001 and issued on Jan. 31,2006, herein incorporated by reference in its entirety, teaches thatmimetics of the N terminal portion of Smac provide viable drugcandidates.

Additionally, it has been shown in U.S. application Ser. No. 10/777,946to McLendon et al. entitled “IAP-Binding Cargo Molecules andPeptidomimetics For Use In Diagnostic and Therapeutic Methods” filed onFeb. 12, 2004, herein incorporated by reference in its entirety, that acargo molecule can be attached to a N-terminal Smac tetrapeptidepeptidomimetic.

SUMMARY OF THE INVENTION

The present invention provides compounds which mimic the tertiarybinding structure of Smac to IAPs or activity of the N-terminal portionof Smac. Stereoisomers of the mimetic compounds described herein arealso encompassed in the present invention. The invention also providesmethods of using these mimetics to modulate apoptosis and further fortherapeutic purposes. The invention also provides intermediates andmethods for using these intermediates for the preparation of compoundswhich modulate apoptosis by mimicking the tertiary binding structure ofSmac to IAPs or activity of the N-terminal portion of Smac.

A compound of the present invention having the general formula (I):

wherein R1 and R2 are independently H, tert-butoxycarbonyl,benzyloxycarbonyl, acetyl, trifluoroacetyl, alkyl,optionally-substituted alkyl, or

where R5a and R5b are independently H, alkyl, cycloalkyl,cycloalkylalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl; or each optionally-substituted with hydroxyl, mercapto,halogen, amino, carboxyl, alkyl, haloalkyl, alkoxy, or alkylthio or R5aand R5b are independently optionally-substituted with hydroxyl,mercapto, halogen, amino, carboxyl, alkyl, haloalkyl, alkoxy, oralkylthio; or, optionally, R5a and R5b are connected by an alkylene,alkenylene, alkynylene bridge of 2 to 12 carbon atoms or anoptionally-substituted alkylene, alkenylene, alkynylene bridge of 2 to12 carbon atoms where one or more carbon atoms are replaced with N, O,or S;R6a and R6b are independently H, tert-butoxycarbonyl, benzyloxycarbonyl,acetyl, trifluoroacetyl, alkyl, lower alkyl, optionally-substitutedalkyl, or

where R7a and R7b are independently H, alkyl, cycloalkyl, haloallyl; orR8a and R7a and R8b and R7b can independently or together form a ringsuch as an aziridine or azetidine ring;R8a and R8b are independently H, hydroxyl, alkyl, aryl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein eachalkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, andheteroarylalkyl is optionally-substituted with halogen, hydroxyl,mercapto, carboxyl, alkyl, alkoxy, amino, and nitro; or R8a and R7a andR8b and R7b can independently or together form a ring such as anaziridine or azetidine ring;R3a and R3b are independently H, halogen, alkyl, aryl, arylalkyl, amino,arylamino, arylalkylamino, hydroxy, alkyloxy, aryloxy, arylalkylhydroxy,dialkylamino, amido, sulfonamido, or amidino;m and n are independently 0, 1, 2, or 3;X and Y are independently O, N, S, or C═C; andR9a, R9b, R10a, R10b are independently H, alkyl, optionally-substitutedalkyl, aryl, heteroaryl, optionally-substituted aryl, heteroaryl, or R9aand R10a, independently or in parallel with R9b and R10b, can be linkedby 4 to 8 optionally-substituted atoms such as C, N, O, or S, to form anaromatic or non-aromatic ring; andwhen Wa and Wb are covalently bound, Wa and Wb are a bond, alkylene,alkenylene, alkynylene, aryl, arylalkylene, arylalkylalkylene,heteroaryl, heteroarylalkylene, or an optionally-substituted alkylene,alkenylene, alkynylene chain of 2 to 12 carbon atoms where one or morecarbon atoms are replaced with N, O, or S; and R11a and R11b areindependently absent, H, alkyl, optionally-substituted alkyl,hydroxyalkyl, alkoxyalkyl; or R11a and R11b together form an alkylene,alkenylene, alkynlyene, or alkyloxyalkylene chain of 2 to 12 carbonatoms where one or more carbon atoms are optionally replaced with N, O,or S;

When Wa and Wb are not covalently bound, Wa and Wb are independently beH, Cl, Br, F, alkyl, CN, CO₂H; and R11a and R11b together form analkylene, alkenylene, alkynylene, or alkyloxyalkylene chain of 2 to 12carbon atoms or an optionally substituted alkylene, alkenylene,alkynylene, or alkyloxyalkylene chain of 2 to 12 carbon atoms where oneor more carbon atoms are optionally replaced with N, O, or S; or Wa isH, Cl, Br, F, alkyl, CN, CO₂H and Wb and R11a together are a bond,alkylene, alkenylene, alkynylene, aryl, arylalkylene, arylalkylalkylene,heteroaryl, heteroarylalkylene, or an optionally-substituted alkylene,alkenylene, alkynylene chain of 2 to 12 carbon atoms where one or morecarbon atoms can be replaced with N, O, or S, and R11b is absent or H,alkyl, optionally-substituted alkyl, hydroxyalkyl, alkoxyalkyl.

Another embodiment of the present invention is the therapeuticcombination of compounds of the present invention with TRAIL or otherchemical or biological agents which bind to and activate the TRAILreceptor(s). TRAIL has received considerable attention recently becauseof the finding that many cancer cell types are sensitive toTRAIL-induced apoptosis, while most normal cells appear to be resistantto this action of TRAIL. TRAIL-resistant cells may arise by a variety ofdifferent mechanisms including loss of the receptor, presence of decoyreceptors, or overexpression of FLIP which competes for zymogencaspase-8 binding during DISC formation. In TRAIL resistance, Smacmimetics increase tumor cell sensitivity to TRAIL leading to enhancedcell death, the clinical correlations of which are expected to beincreased apoptotic activity in TRAIL resistant tumors, improvedclinical response, increased response duration, and ultimately, enhancedpatient survival rate. In support of this, reduction in XIAP levels byin vitro antisense treatment has been shown to cause sensitization ofresistant melanoma cells and renal carcinoma cells to TRAIL(Chawla-Sarkar, et al., 2004). The Smac mimetics disclosed herein bindto IAPs and inhibit their interaction with caspases, thereinpotentiating TRAIL-induced apoptosis.

In another embodiment of the invention, Smac mimetics are used incombination with BCG vaccine treatment of bladder cancer. XIAP, thenominal target of Smac mimetics, is overexpressed in a high proportionof bladder cancers. In studies using antisense XIAP, bladder cancercells were sensitized to chemotherapeutic agents inducing apoptosis ofeffected cells through the TRAIL pathway. The present invention providesSmac mimetics for use with BCG therapy in superficial bladdercancer/carcinoma in situ. The Smac mimetics disclosed herein willenhance the effects of BCG vaccine by enhancing the effects if TRAILgenerated in response to the vaccine.

Similarly, Smac mimetics will augment the TRAIL induced apoptosisobserved in melanoma and renal cell carcinoma patients being treatedwith IL-2. Since IL-2 induces NK cell activity enhancing TRAILexpression, the addition of treatment with a caspase-9 activator, suchas Smac mimetic, will lead to a more efficious clinical response.

Another embodiment of the present invention provides Smac mimetics whichact synergistically with topoismerase inhibitors to potentiate theirapoptotic inducing effect. Topoisomerase inhibitors inhibit DNAreplication and repair, thereby promoting apoptosis and have been usedas chemothemotherapeutic agents. Topoisomerase inhibitors promote DNAdamage by inhibiting the enzymes that are required in the DNA repairprocess. Therefore, export of cytochrome c and Smac from themitochondria into the cell cytosol is induced by the DNA damage causedby topoisomerase inhibitors.

Topoisomerase inhibitors of both the Type I class (camptothecin,topotecan, SN-38, irinotecan, topotecan, BNP 1350, 9-amino-camptothecan,lurtotecan, grimatecan, exatecan, amsacrine, and diflomotecan) and theType II class (etoposide, anthracycyline, anthraquinone, andpodophyllotoxin) show potent synergy with the Smac mimetics of theinvention in a multi-resistant glioblastoma cell line (T98G), breastcancer line (MDA-MB-231), and ovarian cancer line (OVCAR-3) amongothers. Other topoisomerase inhibitors include, for example,Aclacinomycin A, camptothecin, daunorubicin, doxorubicin, ellipticine,epirubicin, and mitaxantrone.

In another embodiment of the invention, thechemotherapeutic/anti-neoplastic agent may be a platinum containingcompound. In one embodiment of the invention the platinum containingcompound is cisplatin. Cisplatin can synergize with a Smacpeptidomimetic and potentiate the inhibition of an IAP, such as but notlimited to XIAP, cIAP-1, c-IAP-2, ML-IAP, etc. In another embodiment aplatinum containing compound is carboplatin. Carboplatin can synergizewith a Smac peptidomimetic and potentiate the inhibition of an IAP,including, but not limited to, XIAP, cIAP-1, c-IAP-2, ML-IAP, etc. Inanother embodiment a platinum containing compound is oxaliplatin. Theoxaliplatin can synergize with a Smac peptidomimetic and potentiate theinhibition of an IAP, including, but not limited to, XIAP, cIAP-1,c-IAP-2, ML-IAP, etc.

In another embodiment of the invention, thechemotherapeutic/anti-neoplastic agent that synergizes with a compoundaccording to the present invention is a taxane. Taxanes areanti-mitotic, mitotic inhibitors or microtubule polymerization agents.Taxanes include but are not limited to, docetaxel and paclitaxel.

Taxanes are characterized as compounds that promote assembly ofmicrotubules by inhibiting tubulin depolymerization, thereby blockingcell cycle progression through centrosomal impairment, induction ofabnormal spindles and suppression of spindle microtubule dynamics. Theunique mechanism of action of taxane is in contrast to other microtubulepoisons, such as Vinca alkaloids, colchicine, and cryptophycines, whichinhibit tubulin polymerization. Microtubules are highly dynamic cellularpolymers made of αβ-tubulin and associated proteins that play key rolesduring mitosis by participating in the organization and function of thespindle, assuring the integrity of the segregated DNA. Therefore, theyrepresent an effective target for cancer therapy.

In another embodiment, any agent that activates the intrinsic apoptoticpathway and/or causes the release of Smac or cytochrome c from themitochondria has the potential to act synergistically with a Smacmimetic.

A combination of a Smac peptidomimetic and a chemotherapeutic/antineoplastic agent and/or radiation therapy of any type that activates theintrinsic or extrinsic pathways or the release of Smac may provide amore effective approach to destroying tumor cells. Smac peptidomimeticsinteract with IAP's, such as XIAP, cIAP-1, cIAP-2, ML-IAP, etc., andblock the IAP mediated inhibition of apoptosis whilechemotherapeutics/anti neoplastic agents and/or radiation therapy killsactively dividing cells by activating the intrinsic apoptotic pathwayleading to apoptosis and cell death. As is described in more detailbelow, embodiments of the invention provide combinations of a Smacpepidomimetc and a chemotherapeutic/anti-neoplastic agent and/orradiation which provide a synergistic action against unwanted cellproliferation. This synergistic action between a Smac peptidomimetic anda chemotherapeutic/anti-neoplastic agent and/or radiation therapy canimprove the efficiency of the chemotherapeutic/anti-neoplastic agentand/or radiation therapy. This will allow for an increase in theeffectiveness of current chemotherapeutic/anti-neoplastic agents orradiation treatment allowing the dose of thechemotherapeutic/anti-neoplastic agent to be lowered, therein providingboth a more effective dosing schedule as well as a more tolerable doseof chemotherapeutic/anti-neoplastic agent and/or radiation therapy.

For simplicity and illustrative purposes, the principles of theinvention are described by referring mainly to an embodiment thereof. Inaddition, in the following description, numerous specific details areset forth in order to provide a thorough understanding of the invention.It will be apparent however, to one of ordinary skill in the art, thatthe invention may be practiced without limitation to these specificdetails. In other instances, well known methods and structures have notbeen described in detail so as not to unnecessarily obscure theinvention.

DESCRIPTION OF THE FIGURES

FIG. 1 is a graph depicting the relative binding affinity of a Smactetrapeptide (AVPI) and a potent Smac mimetic of the present inventionto XIAP BIR-3 using a flourescence polarization assay. Results showed a30,000 fold increase in binding affinity of the Smac mimetic relative tothe Smac tetrapeptide.

FIG. 2 is a graph showing the half life of three Smac mimetics of thepresent invention following a single dose intravenous administration of1 mg/kg in a rat. Results show up to a six hour half-life for themimetics tested.

FIG. 3 is a graph showing the ability of a Smac mimetic of the presentinvention to selectively antagonize proliferation of an ovarian cancercell line SK-OV-3. In this MTT assay, the Smac mimetic displaysanticancer properties at concentrations that have no effect on normaldiploid cell line MRC-5.

FIG. 4 shows the chemopotentiating effect Smac mimetic using melanomacells that have been shown to be resistant to the apoptotic effects ofTRAIL. Assays for cell proliferation revealed that when MDA-MB-231cells, a breast cancer cell line, were treated with a Smacpeptidomimetic of the invention Entry 1 alone the cells were resistantto the antiproliferative effects of the Smac mimetic of the invention.In contrast, when Entry 1 was used in combination with TRAIL there was a1000 fold increase in the antiproliferative effect resulting in a100-fold increase in the cell killing as detected by the correspondingloss in colony formation.

DETAILED DESCRIPTION

It must also be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art. Although anymethods similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the present invention, thepreferred methods are now described. All publications and referencesmentioned herein are incorporated by reference. Nothing herein is to beconstrued as an admission that the invention is not entitled to antedatesuch disclosure by virtue of prior invention.

As used herein, the term “about” means plus or minus 10% of thenumerical value of the number with which it is being used. Therefore,about 50% means in the range of 45%-55%.

“Alkyl” means a branched or unbranched, saturated or unsaturated (i.e.alkenyl, alkynyl) aliphatic hydrocarbon group, having up to 12 carbonatoms unless otherwise specified. When used as part of another term, forexample “alkylamino”, the alkyl portion may be a saturated hydrocarbonchain, however also includes unsaturated hydrocarbon carbon chains suchas“alkenylamino” and “alkynylamino”. Examples of particular alkyl groupsinclude methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl,sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl,n-hexyl, 2-methylpentyl, 2,2-dimethylbutyl, n-heptyl, 3-heptyl,2-methylhexyl, and the like. The terms “lower alkyl” “C1-C4 alkyl” and“alkyl of 1 to 4 carbon atoms” are synonymous and used interchangeablyto mean methyl, ethyl, 1-propyl, isopropyl, cyclopropyl, 1-butyl,sec-butyl or t-butyl. Unless specified, substituted, alkyl groups maycontain one, two, three or four substituents which may be the same ordifferent. Examples of the above substituted alkyl groups include, butare not limited to: cyanomethyl, nitromethyl, hydroxymethyl,trityloxymethyl, propionyloxymethyl, aminomethyl, carboxymethyl,carboxyethyl, carboxypropyl, alkyloxycarbonylmethyl,allyloxycarbonylaminomethyl, carbamoyloxymethyl, methoxymethyl,ethoxymethyl, t-butoxymethyl, acetoxymethyl, chloromethyl, bromomethyl,iodomethyl, trifluoromethyl, 6-hydroxyhexyl, 2,4-dichloro (n-butyl),2-amino (iso-propyl), 2-carbamoyloxyethyl and the like. The alkyl groupmay also be substituted with a carbocycle group. Examples includecyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, andcyclohexylmethyl groups, as well as the corresponding-ethyl, -propyl,-butyl, -pentyl, -hexyl groups, etc. Particular substituted alkyls aresubstituted methyls e.g. a methyl group substituted by the samesubstituents as the “substituted Cn—Cm alkyl” group. Examples of thesubstituted methyl group include groups such as hydroxymethyl, protectedhydroxymethyl (e.g. tetrahydropyranyloxymethyl), acetoxymethyl,carbamoyloxymethyl, trifluoromethyl, chloromethyl, carboxymethyl,bromomethyl and iodomethyl.

“Amino” denotes primary (i.e. —NH2), secondary (i.e. —NRH) and tertiary(i.e. —NRR) amines. Particular secondary and tertiary amines arealkylamine, dialkylamine, arylamine, diarylamine, arylalkylamine anddiarylalkylamine. Particular secondary and tertiary amines aremethylamine, ethylamine, propylamine, isopropylamine, phenylamine,benzylamine dimethylamine, diethylamine, dipropylamine anddisopropylamine.

“Aryl” when used alone or as part of another term means a carbocyclicaromatic group whether or not fused having the number of carbon atomsdesignated or if no number is designated, up to 14 carbon atoms.Particular aryl groups include phenyl, naphthyl, biphenyl,phenanthrenyl, naphthacenyl, and the like (see e.g. Lang's Handbook ofChemistry (Dean, J. A., ed) 13^(th) ed. Table 7-2 [1985]). In aparticular embodiment an aryl group is phenyl. Substituted phenyl orsubstituted aryl denotes a phenyl group or aryl group substituted withone, two, three, four or five substituents chosen, unless otherwisespecified, from halogen (F, Cl, Br, I), hydroxy, protected hydroxy,cyano, nitro, alkyl (such as C1-C6 alkyl), alkoxy (such as C1-C6alkoxy), benzyloxy, carboxy, protected carboxy, carboxymethyl, protectedcarboxymethyl, hydroxymethyl, protected hydroxymethyl, aminomethyl,protected aminomethyl, trifluoromethyl, alkylsulfonylamino,arylsulfonylamino, heterocyclylsulfonylamino, heterocyclyl, aryl, orother groups specified. One or more methyne (CH) and/or methylene (CH2)groups in these substituents may in turn be substituted with a similargroup as those denoted above. Examples of the term “substituted phenyl”includes but is not limited to a mono- or di (halo) phenyl group such as2-chlorophenyl, 2-bromophenyl, 4-chlorophenyl, 2,6-dichlorophenyl,2,5-dichlorophenyl, 3,4-dichlorophenyl, 3-chlorophenyl, 3-bromophenyl,4-bromophenyl, 3,4-dibromophenyl, 3-chloro-4-fluorophenyl,2-fluorophenyl and the like; a mono- or di (hydroxy)phenyl group such as4-hydroxyphenyl, 3-hydroxyphenyl, 2,4-dihydroxyphenyl, theprotected-hydroxy derivatives thereof and the like; a nitrophenyl groupsuch as 3- or 4-nitrophenyl; a cyanophenyl group, for example,4-cyanophenyl; a mono- or di (lower alkyl)phenyl group such as4-methylphenyl, 2,4-dimethylphenyl, 2-methylphenyl,4-(iso-propyl)phenyl, 4-ethylphenyl, 3-(n-propyl)phenyl and the like; amono or di (alkoxy)phenyl group, for example, 3,4-dimethoxyphenyl,3-methoxy-4-benzyloxyphenyl,3-methoxy-4-(1-chloromethyl)benzyloxy-phenyl, 3-ethoxyphenyl,4-(isopropoxy)phenyl, 4-(t-butoxy)phenyl, 3-ethoxy-4-methoxyphenyl andthe like; 3- or 4-trifluoromethylphenyl; a mono- or dicarboxyphenyl or(protected carboxy)phenyl group such 4-carboxyphenyl; a mono- or di(hydroxymethyl)phenyl or (protected hydroxymethyl)phenyl such as3-(protected hydroxymethyl)phenyl or 3,4-di (hydroxymethyl)phenyl; amono- or di (aminomethyl)phenyl or (protected aminomethyl)phenyl such as2-(aminomethyl)phenyl or 2, 4-(protected aminomethyl)phenyl; or a mono-or di (N-(methylsulfonylamino)) phenyl such as3-(N-methylsulfonylamino)) phenyl. Also, the term “substituted phenyl”represents disubstituted phenyl groups where the substituents aredifferent, for example, 3-methyl-4-hydroxyphenyl,3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl,4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl,2-hydroxy-4-chlorophenyl, and the like, as well as trisubstituted phenylgroups where the substituents are different, for example3-methoxy-4-benzyloxy-6-methyl sulfonylamino,3-methoxy-4-benzyloxy-6-phenyl sulfonylamino, and tetrasubstitutedphenyl groups where the substituents are different such as3-methoxy-4-benzyloxy-5-methyl-6-phenyl sulfonylamino. Particularsubstituted phenyl groups are 2-chlorophenyl, 2-aminophenyl,2-bromophenyl, 3-methoxyphenyl, 3-ethoxy-phenyl, 4-benzyloxyphenyl,4-methoxyphenyl, 3-ethoxy-4-benzyloxyphenyl, 3,4-diethoxyphenyl,3-methoxy-4-benzyloxyphenyl,3-methoxy-4-(1-chloromethyl)benzyloxy-phenyl,3-methoxy-4-(1-chloromethyl)benzyloxy-6-methyl sulfonyl aminophenylgroups. Fused aryl rings may also be substituted with the substituentsspecified herein, for example with 1, 2 or 3 substituents, in the samemanner as substituted alkyl groups.

The term alkylene radical as used herein includes reference to adi-functional saturated branched or unbranched hydrocarbon radicalcontaining from 1 to 30 carbon atoms, and includes, for example,methylene (CH₂), ethylene (CH₂CH₂), propylene (CH₂CH₂CH₂),2-methylpropylene (CH₂CH(CH₃)CH₂), hexylene ((CH₂)₆), and the like.Lower alkylene includes an alkylene group of 1 to 10, more preferably 1to 5, carbon atoms.

Substituted alkylene radicals includes reference to a di-functionalsaturated branched or unbranched alkylene radical or group having 1-30carbon atoms and having from 1 to 5 substituents. Lower substitutedalkylene radicals refer to a substituted alkylene radical group, having1-10 carbon atoms, preferably having 1-5 carbon atoms, and having from 1to 5 substituents. Substituents can include but are not limited to thosefor the alkyl groups.

The term alkenyl radical as used herein includes reference to abranched, cyclic hydrocarbon, or unbranched hydrocarbon radical of 2 to30 carbon atoms containing at least one carbon-carbon double bond, suchas ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, t-butenyl,octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl andthe like. The term lower alkenyl includes an alkenyl group of 2 to 10carbon atoms, preferably 2 to 5 carbon atoms, containing at least onecarbon-carbon double bond. The one or more carbon-carbon double bondsmay independently have a cis or trans configuration. Substituted alkenylradical refers to an alkenyl radical or lower alkenyl group having from1 to 5 substituents that can include but are not limited to those forthe alkyl groups.

The term alkenylene radical includes reference to a difunctionalbranched or unbranched hydrocarbon radical or group containing from 2 to30 carbon atoms and at least one carbon-carbon double bond. “Loweralkenylene” includes an alkenylene group of 2 to 10, more preferably 2to 5, carbon atoms, containing one carbon-carbon double bond.Substituted alkenylene radical refers to an alkenylene radical or loweralkenyl group having from 1 to 5 substituents that can include but arenot limited to those for the alkyl groups.

The term alkynyl radical or group refers to straight or branched chainhydrocarbon radical having 2 to 12 carbon atoms and at least one triplebond, some embodiments include alkynyl groups of 2 to 6 carbon atomsthat have one triple bond. A substituted alkynyl will contain one, two,or three substituents as defined for substituted alkyl groups.Alkynylene includes reference to a difunctional branched or unbranchedhydrocarbon chain containing from 2 to 12 carbon atoms and at least onecarbon-carbon triple bond; some embodiments include an alkynylene groupsof 2 to 6 carbon atoms with one triple bond. A substituted alkynylenewill contain one, two, or three substituents as defined for substitutedalkyl groups.

“Heterocyclic group”, “heterocyclic”, “heterocycle”, “heterocyclyl”, or“heterocyclo” alone and when used as a moiety in a complex group such asa heterocycloalkyl group, are used interchangeably and refer to anymono-, bi-, or tricyclic, saturated or unsaturated, aromatic(heteroaryl) or non-aromatic ring having the number of atoms designated,generally from 5 to about 14 ring atoms, where the ring atoms are carbonand at least one heteroatom (nitrogen, sulfur or oxygen). In aparticular embodiment the group incorporates 1 to 4 heteroatoms.Typically, a 5-membered ring has 0 to 2 double bonds and 6- or7-membered ring has 0 to 3 double bonds and the nitrogen or sulfurheteroatoms may optionally be oxidized (e.g. SO, SO₂), and any nitrogenheteroatom may optionally be quaternized. Particular non-aromaticheterocycles include morpholinyl (morpholino), pyrrolidinyl, oxiranyl,oxetanyl, tetrahydrofuranyl, 2,3-dihydrofuranyl, 2H-pyranyl,tetrahydropyranyl, thiiranyl, thietanyl, tetrahydrothietanyl,aziridinyl, azetidinyl, 1-methyl-2-pyrrolyl, piperazinyl andpiperidinyl. A “heterocycloalkyl” group is a heterocycle group asdefined above covalently bonded to an alkyl group as defined above.

Particular 5-membered heterocycles containing a sulfur or oxygen atomand one to three nitrogen atoms include thiazolyl, such as thiazol-2-yland thiazol-2-yl N-oxide, thiadiazolyl such as 1,3,4-thiadiazol-5-yl and1,2,4-thiadiazol-5-yl, oxazolyl such as oxazol-2-yl, and oxadiazolylsuch as 1,3,4-oxadiazol-5-yl, and 1,2,4-oxadiazol-5-yl. Particular5-membered ring heterocycles containing 2 to 4 nitrogen atoms includeimidazolyl such as imidazol-2-yl; triazolyl such as 1,3,4-triazol-5-yl,1,2,3-triazol-5-yl, and 1,2,4-triazol-5-yl, and tetrazolyl such as1H-tetrazol-5-yl. Particular benzo-fused 5-membered heterocycles arebenzoxazol-2-yl, benzthiazol-2-yl and benzimidazol-2-yl. Particular6-membered heterocycles contain one to three nitrogen atoms andoptionally a sulfur or oxygen atom, for example pyridyl, such aspyrid-2-yl, pyrid-3-yl, and pyrid-4-yl; pyrimidyl such as pyrimid-2-yland pyrimid-4-yl; triazinyl such as 1,3,4-triazin-2-yl and1,3,5-triazin-4-yl; pyridazinyl such as pyridazin-3-yl, and pyrazinyl.Substituents for optionally substituted heterocycles, and furtherexamples of the 5- and 6-membered ring systems discussed above can befound in U.S. Pat. No. 4,278,793 to W. Druckheimer et al.

Arylalkyl radical refers to alkyl radicals bearing an aryl substituentand have from about 6 to about 20 carbon atoms (and all combinations andsubcombinations of ranges and specific numbers of carbon atoms therein),with from about 6 to about 12 carbon atoms being preferred. Arylalkylgroups can be optionally substituted. Non-limiting examples include, forexample, benzyl, naphthylmethyl, diphenylmethyl, triphenylmethyl,phenylethyl, and diphenylethyl. A substituted arylalkyl group willcontain one or more substituents on the aryl or alkyl group as definedfor substituted alkyl groups.

Cycloalkylaryl radical or group refers to a cycloalkyl radical fused toan aryl group, including all combinations of independently substitutedalkyl cycloalkylaryls, the cycloalkyl and aryl group having two atoms incommon.

Cycloalkyl radical or group more specifically includes reference to amonovalent saturated carbocyclic alkyl radical consisting of one or morerings in their structures and having from about 3 to about 14 carbonatoms (and all combinations and subcombinations of ranges and specificnumbers of carbon atoms therein), with from about 3 to about 7 carbonatoms being preferred. Multi-ring structures may be bridged or fusedring structures. The rings can optionally be substituted with one ormore of the substituents for the alkyl groups. Examples of cycloalkylgroups include, but are not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclooctyl, and adamantyl. A substitutedcycloalkyl group will contain one or more substituents as defined forsubstituted alkyl groups.

Cycloalkylalkyl radical more specifically refers to alkyl radicalsbearing an cycloalkyl substituent and having from about 4 to about 20carbon atoms (and all combinations and subcombinations of ranges andspecific numbers of carbon atoms therein), with from about 6 to about 12carbon atoms being preferred and can include but are not limited tomethyl-cyclopropyl, methylcyclohexyl, isopropylcyclohexyl, andbutyl-cyclohexyl groups. Cycloalkylalkyl radical or group can beoptionally substituted with one or more substituents for the alkylgroups including but not limited to hydroxy, cyano, alkyl, alkoxy,thioalkyl, halo, haloalkyl, hydroxyalkyl, nitro, amino, alkylamino anddialkylamino.

“Heteroaryl” alone and when used as a moiety in a complex group such asa heteroarylalkyl group, refers to any mono-, bi-, or tricyclic aromaticring system having the number of atoms designated where at least onering is a 5-, 6- or 7-membered ring containing from one to fourheteroatoms selected from the group nitrogen, oxygen, and sulfur (Lang'sHaltdbook of Chemistry, supra). Included in the definition are anybicyclic groups where any of the above heteroaryl rings are fused to abenzene ring. The following ring systems are examples of the heteroaryl(whether substituted or unsubstituted) groups denoted by the term“heteroaryl”: thienyl, furyl, imidazolyl, pyrazolyl, thiazolyl,isothiazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl,oxadiazolyl, tetrazolyl, thiatriazolyl, oxatriazolyl, pyridyl,pyrimidyl, pyrazinyl, pyridazinyl, thiazinyl, oxazinyl, triazinyl,thiadiazinyl, oxadiazinyl, dithiazinyl, dioxazinyl, oxathiazinyl,tetrazinyl, thiatriazinyl, oxatriazinyl, dithiadiazinyl, imidazolinyl,dihydropyrimidyl, tetrahydropyrimidyl, tetrazolo[1,5-b]pyridazinyl andpurinyl, as well as benzo-fused derivatives, for example benzoxazolyl,benzofuryl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl,benzoimidazolyl and indolyl. Particularly“heteroaryls” include;1,3-thiazol-2-yl, 4-(carboxymethyl)-5-methyl-1,3-thiazol-2-yl,4-(carboxymethyl)-5-methyl-1,3-thiazol-2-yl sodium salt,1,2,4-thiadiazol-5-yl, 3-methyl-1,2,4-thiadiazol-5-yl,1,3,4-triazol-5-yl, 2-methyl-1,3,4-triazol-5-yl,2-hydroxy-1,3,4-triazol-5-yl, 2-carboxy-4-methyl-1,3,4-triazol-5-ylsodium salt, 2-carboxy-4-methyl-1,3,4-triazol-5-yl, 1,3-oxazol-2-yl,1,3,4-oxadiazol-5-yl, 2-methyl-1,3,4-oxadiazol-5-yl,2-(hydroxymethyl)-1,3,4-oxadiazol-5-yl, 1,2,4-oxadiazol-5-yl,1,3,4-thiadiazol-5-yl, 2-thiol-1,3,4-thiadiazol-5-yl,2-(methylthio)-1,3,4-thiadiazol-5-yl, 2-amino-1,3,4-thiadiazol-5-yl,1H-tetrazol-5-yl, 1-methyl-1H-tetrazol-5-yl, 1-(1-(dimethylamino)eth-2-yl)-1H-tetrazol-5-yl, 1-(carboxymethyl)-1H-tetrazol-5-yl,1-(carboxymethyl)-1H-tetrazol-5-yl sodium salt, 1-(methylsulfonicacid)-1H-tetrazol-5-yl, 1-(methylsulfonic acid)-1H-tetrazol-5-yl sodiumsalt, 2-methyl-1H-tetrazol-5-yl, 1,2,3-triazol-5-yl,1-methyl-1,2,3-triazol-5-yl, 2-methyl-1,2,3-triazol-5-yl,4-methyl-1,2,3-triazol-5-yl, pyrid-2-yl N-oxide,6-methoxy-2-(n-oxide)-pyridaz-3-yl, 6-hydroxypyridaz-3-yl,1-methylpyrid-2-yl, 1-methylpyrid-4-yl, 2-hydroxypyrimid-4-yl,1,4,5,6-tetrahydro-5,6-dioxo-4-methyl-as-triazin-3-yl,1,4,5,6-tetrahydro-4-(formylmethyl)-5,6-dioxo-as-triazin-3-yl,2,5-dihydro-5-oxo-6-hydroxy-astriazin-3-yl,2,5-dihydro-5-oxo-6-hydroxy-as-triazin-3-yl sodium salt,2,5-dihydro-5-oxo-6-hydroxy-2-methyl-astriazin-3-yl sodium salt,2,5-dihydro-5-oxo-6-hydroxy-2-methyl-as-triazin-3-yl,2,5-dihydro-5-oxo-6-methoxy-2-methyl-as-triazin-3-yl,2,5-dihydro-5-oxo-as-triazin-3-yl,2,5-dihydro-5-oxo-2-methyl-as-triazin-3-yl,2,5-dihydro-5-oxo-2,6-dimethyl-as-triazin-3-yl,tetrazolo[1,5-b]pyridazin-6-yl and8-aminotetrazolo[1,5-b]-pyridazin-6-yl. An alternative group of“heteroaryl” includes; 4-(carboxymethyl)-5-methyl-1,3-thiazol-2-yl,4-(carboxymethyl)-5-methyl-1,3-thiazol-2-yl sodium salt,1,3,4-triazol-5-yl, 2-methyl-1,3,4-triazol-5-yl, 1H-tetrazol-5-yl,1-methyl-1H-tetrazol-5-yl, 1-(1-(dimethylamino)eth-2-yl)-1H-tetrazol-5-yl, 1-(carboxymethyl)-1H-tetrazol-5-yl,1-(carboxymethyl)-1H-tetrazol-5-yl sodium salt, 1-(methylsulfonicacid)-1H-tetrazol-5-yl, 1-(methylsulfonic acid)-1H-tetrazol-5-yl sodiumsalt, 1,2,3-triazol-5-yl,1,4,5,6-tetrahydro-5,6-dioxo-4-methyl-as-triazin-3-yl,1,4,5,6-tetrahydro-4-(2-formylmethyl)-5,6-dioxo-as-triazin-3-yl,2,5-dihydro-5-oxo-6-hydroxy-2-methyl-as-triazin-3-yl sodium salt,2,5-dihydro-5-oxo-6-hydroxy-2-methyl-as-triazin-3-yl,tetrazolo[1,5-b]pyridazin-6-yl, and8-aminotetrazolo[1,5-b]pyridazin-6-yl.

“Inhibitor” means a compound which reduces or prevents the binding ofIAP proteins to caspase proteins or which reduces or prevents theinhibition of apoptosis by an IAP protein, or which binds to an IAP BIRdomain in a manner similar to the amino terminal portion of Smac,thereby freeing Smac to inhibit the action of an IAP.

“Pharmaceutically acceptable salts” include both acid and base additionsalts. “Pharmaceutically acceptable acid addition salt” refers to thosesalts which retain the biological effectiveness and properties of thefree bases and which are not biologically or otherwise undesirable,formed with inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, carbonic acid, phosphoric acid and the like,and organic acids may be selected from aliphatic, cycloaliphatic,aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes oforganic acids such as formic acid, acetic acid, propionic acid, glycolicacid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid,maleic acid, maloneic acid, succinic acid, fumaric acid, tartaric acid,citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilicacid, benzoic acid, cinnamic acid, mandelic acid, embonic acid,phenylacetic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicyclic acid and the like.

The terms “mimetic,” “peptide mimetic” and “peptidomimetic” are usedinterchangeably herein, and generally refer to a peptide, partialpeptide or non-peptide molecule that mimics the tertiary bindingstructure or activity of a selected native peptide or protein functionaldomain (e.g., binding motif or active site). These peptide mimeticsinclude recombinantly or chemically modified peptides, as well asnon-peptide agents such as small molecule drug mimetics, as furtherdescribed below.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, rash, or gastric upset.

“Providing” when used in conjunction with a therapeutic means toadminister a therapeutic directly into or onto a target tissue or toadminister a therapeutic to a patient whereby the therapeutic positivelyimpacts the tissue to which it is targeted.

As used herein “subject” or “patient” refers to an animal or mammalincluding, but not limited to, human, dog, cat, horse, cow, pig, sheep,goat, chicken, monkey, rabbit, rat, mouse, etc.

As used herein, the term “therapeutic” means an agent utilized to treat,combat, ameliorate, prevent or improve an unwanted condition or diseaseof a patient. Embodiments of the present invention are directed topromote apoptosis, and thus cell death.

The terms “therapeutically effective amount” or “effective amount”, asused herein, may be used interchangeably and refer to an amount of atherapeutic compound component of the present invention. For example, atherapeutically effective amount of a therapeutic compound is apredetermined amount calculated to achieve the desired effect, i.e., toeffectively promote apoptosis, or to sensitize a cell to apoptosispreferably by eliminating an IAP inhibition of apoptosis, morepreferably by inhibiting an IAP binding to a caspase.

“Mimetics” or “peptidomimetics” are synthetic compounds having athree-dimensional structure (i.e. a “core peptide motif”) based upon thethree-dimensional structure of a selected peptide. The peptide motifprovides the mimetic compound with the desired biological activity,i.e., binding to IAP, wherein the binding activity of the mimeticcompound is not substantially reduced, and is often the same as orgreater than the binding affinity of the native peptide on which themimetic is modeled. For example, in the mimetics of the presentinvention, we have found that X₃ and X₄ can be quite non-peptide like.Peptidomimetic compounds can have additional characteristics thatenhance their therapeutic application, such as increased cellpermeability, greater affinity and/or avidity and prolonged biologicalhalf-life.

Mimetic, specifically, peptidomimetic design strategies are readilyavailable in the art and can be easily adapted for use in the presentinvention (see, e.g., Ripka & Rich, Curr. Op. Chem. Biol. 2, 441-452,1998; Hruby et al., Curr. Op. Chem. Biol. 1, 114-119, 1997; Hruby &Balse, Curr. Med. Chem. 9, 945-970, 2000). One class of mimetic mimics abackbone that is partially or completely non-peptide, but mimics thepeptide backbone atom-for-atom and comprises side groups that likewisemimic the functionality of the side groups of the native amino acidresidues. Several types of chemical bonds, e.g. ester, thioester,thioamide, retroamide, reduced carbonyl, dimethylene and ketomethylenebonds, are known in the art to be generally useful substitutes forpeptide bonds in the construction of protease-resistant peptidomimetics.Another class of peptidomimetics comprises a small non-peptide moleculethat binds to another peptide or protein, but which is not necessarily astructural mimetic of the native peptide. Yet another class ofpeptidomimetics has arisen from combinatorial chemistry and thegeneration of massive chemical libraries. These generally comprise noveltemplates which, though structurally unrelated to the native peptide,possess necessary functional groups positioned on a nonpeptide scaffoldto serve as “topographical” mimetics of the original peptide (Ripka &Rich, 1998, supra). Tetrapeptidomimetics of the invention are of thetype disclosed and claimed in U.S. Pat. No. 6,992,063 to Shi et al.

It has been demonstrated in accordance with the present invention thatthe IAP-binding peptides or mimetics thereof are capable of potentiatingapoptosis of cells.

Mimetics of the core IAP-binding portions are preferred. The mimeticsdescribed herein are suitably small, and since structural features inrelation to the IAP binding groove are well-characterized, a widevariety of mimetic compounds may be synthesized. Added advantages ofcompounds of this size include improved solubility in aqueous solutionand ease of delivery to selected targets in vivo.

In one embodiment, the IAP-binding peptides of the invention aremodified to produce peptide mimetics by replacement of one or morenaturally occurring side chains of the 20 genetically encoded aminoacids, or D amino acids with other side chains, for instance with groupssuch as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl,amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy,carboxy and the lower ester derivatives thereof, and with 4-, 5-, 6-, to7-membered heterocyclics. For example, proline analogs can be made inwhich the ring size of the proline residue is changed from 5 members to4, 6, or 7 members. Cyclic groups can be saturated or unsaturated, andif unsaturated, can be aromatic or non-aromatic. Heterocyclic groups cancontain one or more nitrogen, oxygen, and/or sulphur heteroatoms.Examples of such groups include the furazanyl, imidazolidinyl,imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g.morpholino), oxazolyl, piperazinyl (e.g. 1-piperazinyl), piperidyl (e.g.1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl,pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl(e.g. 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl,thienyl, thiomorpholinyl (e.g. thiomorpholino), and triazolyl. Theseheterocyclic groups can be substituted or unsubstituted. Where a groupis substituted, the substituent can be alkyl, alkoxy, halogen, oxygen,or substituted or unsubstituted phenyl. Peptidomimetics may also haveamino acid residues that have been chemically modified byphosphorylation, sulfonation, biotinylation, or the addition or removalof other moieties.

The present invention provides compounds which mimic the tertiarybinding structure of Smac to IAPs or activity of the N-terminal portionof Smac. Stereoisomers of the mimetic compounds described herein arealso encompassed in the present invention. The invention also providesmethods of using these mimetics to modulate apoptosis and further fortherapeutic purposes. The invention also provides intermediates andmethods for using these intermediates for the preparation of compoundswhich modulate apoptosis by mimicking the tertiary binding structure ofSmac to IAPs or activity of the N-terminal portion of Smac.

In accordance with the present invention, a compound of the presentinvention having the general formula (I) is provided:

wherein R1 and R2 are independently H, tert-butoxycarbonyl,benzyloxycarbonyl, acetyl, trifluoroacetyl, alkyl,optionally-substituted alkyl, or

where R5a and R5b are independently H, alkyl, cycloalkyl,cycloalkylalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl or each optionally-substituted with hydroxyl, mercapto,halogen, amino, carboxyl, alkyl, haloalkyl, alkoxy, or alkylthio; or R5aand R5b are independently optionally-substituted with hydroxyl,mercapto, halogen, amino, carboxyl, alkyl, haloalkyl, alkoxy, oralkylthio; or, optionally, R5a and R5b are connected by an alkylene,alkenylene, alkynylene bridge of 2 to 12 carbon atoms oroptionally-substituted alkylene, alkenylene, alkynylene bridge of 2 to12 carbon atoms where one or more carbon atoms are replaced with N, O,or S;R6a and R6b are independently H, tert-butoxycarbonyl, benzyloxycarbonyl,acetyl, trifluoroacetyl, alkyl, lower alkyl, optionally-substitutedalkyl, or

where R7a and R7b are independently H, alkyl, cycloalkyl, haloalkyl; orR8a and R7a and R8b and R7b can independently or together form a ringsuch as an aziridine or azetidine ring;R8a and R8b are independently H, hydroxyl, alkyl, aryl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein eachalkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, andheteroarylalkyl is optionally-substituted with halogen, hydroxyl,mercapto, carboxyl, alkyl, alkoxy, amino, and nitro; or R8a and R7a andR8b and R7b can independently or together form a ring such as anaziridine or azetidine ring;R3a and R3b are independently H, halogen, alkyl, aryl, arylalkyl, amino,arylamino, arylalkylamino, hydroxy, alkyloxy, aryloxy, arylalkylhydroxy,dialkylamino, amido, sulfonamido, or amidino;m and n are independently 0, 1, 2, or 3;X and Y are independently O, N, S, or C═C;R9a, R9b, R10a, R10b are independently H, alkyl, optionally-substitutedalkyl, aryl, heteroaryl, optionally-substituted aryl, heteroaryl, or R9aand R10a, independently or in parallel with R9b and R10b, can be linkedby 4 to 8 optionally-substituted atoms such as C, N, O, or S, to form anaromatic or non-aromatic ring;When Wa and Wb are covalently bound, Wa and Wb are a bond, alkylene,alkenylene, alkynylene, aryl, arylalkylene, arylalkylalkylene,heteroaryl, heteroarylalkylene, or an optionally-substituted alkylene,alkenylene, alkynylene chain of 2 to 12 carbon atoms where one or morecarbon atoms can be replaced with N, O, or S; and R11a and R11b areindependently absent, H, alkyl, optionally-substituted alkyl,hydroxyalkyl, alkoxyalkyl; or R11a and R11b together form an alkylene,alkenylene, alkynlyene, or alkyloxyalkylene chain of 2 to 12 carbonatoms where one or more carbon atoms are, optionally, replaced with N,O, or S;When Wa and Wb are not covalently bound, Wa and Wb are independently beH, Cl, Br, F, alkyl, CN, CO2H; and R11a and R11b together form analkylene, alkenylene, alkynylene, or alkyloxyalkylene chain of 2 to 12carbon atoms or optionally substituted alkylene, alkenylene, alkynylene,or alkyloxyalkylene chain of 2 to 12 carbon atoms where one or morecarbon atoms can be replaced with N, O, or S; or Wa can be H, Cl, Br, F,alkyl, CN, CO₂H and Wb and R11a together are a bond, alkylene,alkenylene, alkynylene, aryl, arylalkylene, arylalkylalkylene,heteroaryl, heteroarylalkylene, or an optionally-substituted alkylene,alkenylene, alkynylene chain of 2 to 12 carbon atoms where one or morecarbon atoms can be replaced with N, O, or S; and R11b is absent or H,alkyl, optionally-substituted alkyl, hydroxyalkyl, alkoxyalkyl.

The compounds encompassed in the present invention include both Smacmimetics and intermediates therefor. The present invention includesstereosiomers of each disclosed compound. Generally, the compounds ofthe present invention include tertrapetide mimetics of Smac, covelentlyattached dimers of tetrapeptide mimetics of Smac and covelently attachedhomodimers of tetrapeptide mimetics of Smac. Homodimers are thosemimetics wherein the substantially identical tetrapeptide mimetics arecovelently bound.

The experimental schemes below are related to the schemes used toproduce the compounds first disclosed in PCT Publication No. WO2004/007529, herein incorporated by reference in its entirety. Suitablepeptides and peptidomimetics are also described in U.S. application Ser.No. 11/184,503 filed Jul. 15, 2005, entitled “IAP Binding Compounds”based upon U.S. Provisional Application No. 60/588,050 filed Jul. 15,2004, the disclosures of which are herein incorporated by reference intheir entireties.

The binding affinity of compounds embodies in the present invention tothe XIAP was determined as described by Nikolovska-Coleska, Z. et. al.(Analytical Biochemistry (2004), vol. 332:261-273) using a variety offluorogenic substrates and is reported as a K_(D) value. Briefly,various concentrations of test peptides were mixed with 5 nMfluorescently labeled peptide (AbuRPF-K(5-Fam)-NH2) and 40 nM ofXIAP-BIR3 for 15 min at RT in 100 μL of 0.1M Potassium Phosphate buffer,pH 7.5 containing 100 μg/ml bovine γ-globulin. Following incubation, thepolarization values (mP) were measured on a Victor²V using a 485 nmexcitation filter and a 520 nm emission filter. IC₅₀ values weredetermined from the plot using nonlinear least-squares analysis usingGraphPad Prism. The compounds described herein afford K_(D) values inthe ranges of K_(D)<0.1 μM (A), K_(D)=0.1-1 μM (B), K_(D)=1-10 μM (C),and K_(D)>10 μM (D).

2-(2-Bromo-6-fluoro-1H-indol-3-ylmethyl)-pyrrolidine-1-carboxylic acidtert-butyl ester (2)

NBS (3.2 g, 17.9 mmol) was added to a solution containing 1 (5.4 g, 17.0mmol) in CCl₄ (50 mL). The heterogeneous reaction mixture was heated toreflux (80-85° C.) for 2 h at which point TLC analysis revealed completeconsumption of 1. [TLC analysis, 4:1 hexane/EtOAc, R_(f)(8)=0.4;R_(f)(2)=0.5]. The reaction mixture was cooled to ambient temperaturethen poured onto a column of silica gel. The product was eluted with10-15% EtOAc/hexane to afford 4.4 g (65%) of 2 as a white solid. ¹H NMR(DMSO, 300 MHz) δ11.74 (s, 1H), 7.56 (m, 1H), 7.02 (d, J=9.3 Hz, 1H),6.88 (m, 1H), 3.99 (m, 1H), 3.22 (m, 2H), 2.97 (m, 1H), 2.58 (dd,J=13.5, 9.3 Hz, 1H), 1.9-1.5 (4H), 1.40 (s, 9H) ppm.

1,4-Bis-[2-(6-Fluoro-1H-indol-3-ylmethyl)-pyrrolidine-1-carboxylic acidtert-butyl ester]benzene (4)

A solution containing 2 (3.3 g, 8.3 mmol) in toluene (25 mL), EtOH (25mL), and water (1 mL) was degassed under high vacuum. K₂CO₃ (4.5 g, 32.5mmol), 3 (0.97 g, 5.8 mmol), and (Ph₃P)₄Pd (0.29 g, 0.25 mmol) wereadded and the resulting mixture was stirred at 100° C. for 5 h. [TLCanalysis, 4:1 hexane/EtOAc, R_(f)(2)=0.5; R_(f)(4)=0.3]. The reactionmixture was filtered through a short pad of silica gel and washed with5% EtOAc/hexane. The filtrate was concentrated and the crude product waspurified by flash silica gel chromatography (20% EtOAc/hexane) to afford3.0 g (98%) of 4 as an off-white, highly-fluorescent solid. ¹H NMR(CDCl₃, 300 MHz) δ8.6-8.4 (m, 2H), 7.65 (m, 2H), 7.57 (br s, 4H), 7.05(m, 2H), 7.90 (m, 2H), 4.22 (br s, 2H), 3.4-3.1 (m, 6H), 2.90 (m, 2H),1.8-1.3 (m, 26H) ppm.

1,4-Bis-{2-[1-(2-Acetoxy-ethyl)-6-fluoro-1H-indol-3-ylmethyl]-pyrrolidine-1-carboxylicacid tert-butyl ester}benzene (6)

To a suspension of 60% NaH (0.67 g, 17.0 mmol) in anhydrous DMF (10 mL)was added a solution of 4 (3.0 g, 4.2 mmol) in DMF (10 mL) at 0° C. Thereaction mixture was allowed to stir at ambient temperature for 1 h thenre-cooled to 0° C. A solution containing 5 (2.8 g, 16.8 mmol) in DMF (5mL) was added to the reaction mixture and the ice bath was removedfollowing addition. After 2 h at ambient temperature, LC/MS and TLCanalyses revealed complete consumption of 4. [TLC analysis, 2:1hexane/EtOAc, R_(f)(4)=0.4; R_(f)(6)=0.8]. The reaction mixture wascooled to 0° C. and saturated aqueous NH₄Cl was added. The product wasextracted with diethyl ether. The ether extracts were washed with water,brine, dried over anhydrous Na₂SO₄, filtered, and concentrated. Thecrude product was purified by NP—HPLC (silica gel, 10-100% EtOAc/hexaneover 30 min) to afford 1.4 g of 6 as an off-white solid. ¹H NMR (CDCl₃,300 MHz) δ7.68 (m, 2H), 7.54 (s, 4H), 7.12 (m, 2H), 6.94 (m, 2H), 4.25(m, 4H), 4.14 (m, 6H), 3.4-3.1 (6H), 2.60 (dd, J=9.6, 13.8 Hz, 2H), 1.90(s, 6H), 1.83 (m, 2H), 1.7-1.3 (m, 24H) ppm.

Acetic acid2-(2-{4-[1-(2-acetoxy-ethyl)-6-fluoro-3-pyrrolidin-2-ylmethyl-1H-indol-2-yl]-phenyl}-6-fluoro-3-pyrrolidin-2-ylmethyl-indol-1-yl)-ethylester (7)

A solution containing 6 (1.4 g, 1.58 mmol) in DCM (20 mL) was cooled to0° C. TFA (5 mL) was added via pipette and the reaction was allowed towarm to ambient temperature and monitored until TLC analysis revealedcomplete consumption of 6 (˜2 h). TLC analysis, 10% MeOH/DCM,R_(f)(6)=0.7; R_(f)(7)=0.2. The solvent was removed on a rotaryevaporator and the residue was dissolved in EtOAc. The EtOAc solutionwas washed twice with saturated aqueous NaHCO3 and once with brine. Thecombined aqueous washes were back-extracted with EtOAc and the organicextracts were dried over anhydrous Na2SO4, filtered, and concentrated toafford 1.2 g (quant.) of 7 as a yellow solid which was used withoutfurther purification. 1H NMR (CDCl₃, 300 MHz) δ8.05 (dd, J=8.4, 5.4 Hz,2H), 7.56 (s, 4H), 7.13 (dd, J=9.9, 2.4 Hz, 2H), 6.99 (m, 2H), 4.60 (d,J=9.9 Hz, 2H), 4.51 (m, 2H), 4.26 (m, 4H), 4.15 (m, 4H), 3.63 (m, 2H),3.54 (m, 2H), 3.5-3.3 (m, 4H), 2.41 (m, 2H), 1.89 (s, 6H), 1.8-1.5 (m,6H), 1.43 (s, 18H), 1.09 (s, 18H) ppm.

1,4-Bis-{Acetic acid2-{3-[1-(2-tert-butoxycarbonylamino-3,3-dimethyl-butyryl)-pyrrolidin-2-ylmethyl]-6-fluoro-indol-1-yl}-ethylester}benzene (8)

A solution containing Boc-L-tert-Leu-OH (0.82 g, 3.54 mmol) and HATU(1.41 g, 3.70 mmol) in anhydrous NMP (15 mL) was cooled to 0° C. After15 min, N-methylmorpholine (0.46 g, 0.5 mL, 4.54 mmol) was added viasyringe. After 15 min, a solution containing 7 (1.10 g, 1.61 mmol) inDCM (10 mL) was added and the reaction mixture was allowed to warm toambient temperature over 16 h at which point TLC analysis revealedcomplete consumption of 7 [TLC analysis, 2:1 hexane/EtOAc,R_(f)(7)=0.01; R_(f)(8)=0.8]. The reaction mixture was diluted withdiethyl ether and washed once with dilute aqueous HCl, five times withwater to remove excess NMP, once with saturated aqueous NaHCO₃ andbrine, dried over anhydrous Na₂SO₄, filtered, and concentrated. Thecrude product was purified by NP—HPLC (silica gel, 10-100% EtOAc/hexaneover 30 min) to afford 1.3 g (73%) of 8 as an off-white solid. ¹H NMR(CDCl₃, 300 MHz) δ8.05 (dd, J=5.4, 8.4 Hz, 2H), 7.56 (s, 4H), 7.11 (dd,J=2.4, 9.9 Hz, 2H), 6.98 (m, 2H), 5.43 (d, J=9.9 Hz, 2H), 4.51 (m, 2H),4.26 (m, 6H), 4.17 (m, 6H), 3.2-3.7 (m, 8H), 2.41 (dd, J=12, 13 Hz, 2H),1.88 (s, 6H), 1.7-1.5 (m, 4H), 1.43 (s, 18H), 1.04 (s, 18H) ppm.

Acetic acid2-{2-(4-{1-(2-acetoxy-ethyl)-3-[1-(2-amino-3,3-dimethyl-butyryl)-pyrrolidin-2-ylmethyl]-6-fluoro-1H-indol-2-yl}-phenyl)-3-[1-(2-amino-3,3-dimethyl-butyryl)-pyrrolidin-2-ylmethyl]-6-fluoro-indol-1-yl}-ethylester (9)

A solution containing 8 (1.3 g, 1.17 mmol) in DCM (5 mL) was cooled to0° C. 20% TFA in DCM (25 mL) was added via pipette and the reaction wasallowed to warm to ambient temperature and monitored until TLC analysisrevealed complete consumption of 8 (˜2 h). TLC analysis, 10% MeOH/DCM,R_(f)(8)=0.7; R_(f)(9)=0.3. The solvent was removed on a rotaryevaporator and the residue was purified by RP-HPLC (Method: Solvent A:water w/0.1% v/v HOAc, Solvent B: ACN w/0.1% v/v HOAc. Dynamax MicrosorbC18 60 Å 8μ, 41.4 mm×25 cm; Flow: 40 mL/min; Detector: 254 nm). Theproduct-containing fractions were pooled and neutralized with saturatedaqueous NaHCO₃. The product was extracted with EtOAc and the organicextract was washed with brine, dried over anhydrous Na₂SO₄, filtered,and concentrated to afford 0.80 g (75%) of 9 as an off-white solid. ¹HNMR (CDCl₃, 300 MHz) δ8.09 (dd, J=5.1, 8.7 Hz, 2H), 7.51 (s, 4H), 7.13(m, 2H), 7.0 (m, 2H), 4.41 (m, 2H), 4.25 (m, 4H), 4.16 (m, 4H), 3.6-3.0(m, 6H), 2.86 (m, 2H), 2.39 (m, 2H), 1.91 (s, 6H), 1.8-1.4 (m, 12H),1.04 (s, 18H) ppm.

1,4-Bis-{Acetic acid2-[3-(1-{2-[2-(tert-butoxycarbonyl-methyl-amino)-propionylamino]-3,3-dimethyl-butyryl}-pyrrolidin-2-ylmethyl)-6-fluoro-indol-1-yl]-ethylester}benzene (10)

A solution containing Boc-L-N(Me)Ala-OH (0.27 g, 1.32 mmol) and HATU(0.54 g, 1.43 mmol) in anhydrous NMP (15 mL) was cooled to 0° C. After15 min, N-methylmorpholine (0.17 g, 0.2 mL, 1.68 mmol) was added viasyringe. After 15 min, a solution containing 9 (0.50 g, 0.55 mmol) inDCM (10 mL) was added and the reaction mixture was allowed to warm toambient temperature over 16 h at which point TLC analysis revealedcomplete consumption of 9 [TLC analysis, 3:2 hexane/EtOAc,R_(f)(9)=0.01; R_(f)(10)=0.5]. The reaction mixture was diluted withdiethyl ether and washed once with dilute aqueous HCl, five times withwater to remove excess NMP, once with saturated aqueous NaHCO₃ andbrine, dried over anhydrous Na₂SO₄, filtered, and concentrated. Thecrude product was purified by NP—HPLC (silica gel, 10-100% EtOAc/hexaneover 30 min) to afford 0.64 g (91%) of 10 as an off-white solid. ¹H NMR(CDCl₃, 300 MHz) δ8.05 (m, 2H), 7.58 (br s, 4H), 7.13 (m, 2H), 6.97 (m,2H), 4.75 (m, 2H), 4.60 (d, J=9.3 Hz, 2H), 4.50 (m, 2H), 4.25 (m, 4H),4.16 (m, 4H), 3.70 (m, 2H), 3.57 (m, 2H), 3.5-3.2 (m, 4H), 2.85 (br s,6H), 2.42 (m, 2H), 1.88 (s, 6H), 1.8-1.4 (m, 8H), 1.52 (s, 18H), 1.33(m, 6H), 1.04 (br s, 18H) ppm.

1,4-Bis-{Acetic acid2-(3-{1-[3,3-dimethyl-2-(2-methylamino-propionylamino)-butyryl]-pyrrolidin-2-ylmethyl}-6-fluoro-indol-1-yl)-ethylester}benzene (11)

A solution containing 10 (0.64 g, 0.5 mmol) in DCM (20 mL) was cooled to0° C. TFA (5 mL) was added via pipette and the reaction was allowed towarm to ambient temperature and monitored until TLC analysis revealedcomplete consumption of 10 (˜2 h). The solvent was removed on a rotaryevaporator and the residue was purified by RP-HPLC (Method: Solvent A:water w/0.1% v/v HOAc, Solvent B: ACN w/0.1% v/v HOAc. Dynamax MicrosorbC18 60 Å 8μ, 41.4 mm×25 cm; Flow: 40 mL/min; Detector: 254 nm). Theproduct-containing fractions were pooled and neutralized with saturatedaqueous NaHCO₃. The product was extracted with EtOAc and the organicextract was washed with brine, dried over anhydrous Na₂SO₄, filtered,and concentrated to afford 0.50 g (93%) of 11 as an off-white solid. ¹HNMR (CDCl₃, 300 MHz) δ8.04 (m, 2H), 7.83 (d, J=9.3 Hz, 2H), 7.55 (m,4H), 7.12 (m, 2H), 6.99 (m, 2H), 4.60 (d, J=9.3 Hz, 2H), 4.57 (m, 2H),4.24 (m, 4H), 3.73 (m, 2H), 3.55 (m, 2H), 3.41 (m, 2H), 3.30 (m, 2H),3.08 (m, 2H), 2.40 (s, 6H), 2.38 (m, 2H), 1.87 (s, 6H), 1.8-1.3 (m,16H), 1.04 (br s, 18H) ppm.

1,4-Bis-{N-(1-{2-[6-Fluoro-1-(2-hydroxy-ethyl)-1H-indol-3-ylmethyl]-pyrrolidine-1-carbonyl}-2,2-dimethyl-propyl)-2-methylamino-propionamide}benzene(12)

Aqueous NaOH (1 M, 5 mL, excess) was added at 0° C. to a solutioncontaining 11 (0.48 g, 0.44 mmol) in MeOH (5 mL). Following theaddition, the ice bath was removed and the reaction mixture was stirredat ambient temperature for 1 h. The reaction mixture was diluted withwater/EtOAc and the layers were separated. The organic phase was washedwith brine, dried over anhydrous Na₂SO₄, filtered, and concentrated. Theresidue was purified by RP-HPLC (Method: Solvent A: water w/0.1% v/vHOAc, Solvent B: ACN w/0.1% v/v HOAc. Dynamax Microsorb C18 60 Å 8μ,41.4 mm×25 cm; Flow: 40 mL/min; Detector: 254 nm). Theproduct-containing fractions were pooled, frozen, and lyophilized toafford 0.19 g of 12 as a flocculent, white solid. ¹H NMR (CDCl₃, 300MHz) δ ppm. ¹³C NMR (CDCl₃, 75 MHz) δ7.8-7.4 (m, 8H), 7.11 (m, 2H), 6.95(m, 2H), 4.57 (d, J=9.3 Hz, 2H), 4.4-4.0 (m, 6H), 3.8-3.4 (m, 8H),3.2-3.0 (m, 3H), 2.6-2.4 (m, 14H), 2.38 (m, 6H), 2.2-1.5 (m, 12H), 1.29(d, J=6.9 Hz, 6H), 1.00 (s, 18H) ppm.

EXAMPLES

Example 1

where R7a and R7b are independently H, alkyl, cycloalkyl, haloalkyl; orR8a and R7a and R8b and R7b can independently or together form a ringsuch as an aziridine or azetidine ring;

R8a and R8b are independently H, hydroxyl, alkyl, aryl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein eachalkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, andheteroarylalkyl is optionally-substituted with halogen, hydroxyl,mercapto, carboxyl, alkyl, alkoxy, amino, and nitro; or R8a and R7a andR8b and R7b can independently or together form a ring such as anaziridine or azetidine ring;

where R5a and R5b are independently H, alkyl, cycloalkyl,cycloalkylalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl; or each optionally-substituted with hydroxyl, mercapto,halogen, amino, carboxyl, alkyl, haloalkyl, alkoxy, or alkylthio; or, insome instances, the R5a and R5b residues are connected by an alkylene,alkenylene, alkynylene bridge of 2 to 12 carbon atoms oroptionally-substituted alkylene, alkenylene, alkynylene bridge of 2 to12 carbon atoms where one or more carbon atoms can be replaced with N,O, or S;

R12a, R12b, R13a, R13b, R14a, and R14b are independently H, Cl, Br, F,alkyl, cycloalkyl, hydroxy, alkoxy, amino, alkylamino, cyano, or CO₂H;

R3a and R3b are independently H, halogen, alkyl, aryl, arylalkyl, amino,arylamino, arylalkylamino, hydroxy, alkyloxy, aryloxy, arylalkylhydroxy,dialkylamino, amido, sulfonamido, or amidino;

X and Y are independently O, N, S, or C═C;

R11a and R11b are absent or independently H, alkyl,optionally-substituted alkyl, hydroxyalkyl, alkoxyalkyl; or R11a andR11b together form an alkylene, alkenylene, alkynlyene, oralkyloxyalkylene chain of 2 to 12 carbon atoms where one or more carbonatoms can be replaced with N, O, or S;

Wa and Wb together are a bond, alkylene, alkenylene, alkynylene, aryl,heteroaryl, or an optionally-substituted alkylene, alkenylene,alkynylene chain of 2 to 12 carbon atoms where one or more carbon atomscan be replaced with N, O, or S.

Stereochem at Position Entry R8a R7a R5a (*) X Y R11 Wa-Wb 1 H S-MeS—iPr S O O na 1,4-phenyl 2 H S-Me S—iPr S O O na trans- (CH═CH) 3 HS-Me S—iPr S O O na CH₂CH₂ 4 H S-Me S—iPr S O O na 1,4-phenyl 6 H S-MeS—iPr S N N NH 1,4-phenyl 7 H S-Me S-tBu S O O na 1,4-phenyl 8 Me S-MeS-tBu S O O na 1,4-phenyl 9 H S-Et S-tBu S O O na 1,4-phenyl 10 Me S-MeS—iPr S O O na 1,4-phenyl 11 H S-Et S—iPr S O O na 1,4-phenyl 12 H S-MeS-cHex S O O na 1,4-phenyl 13 Me S-Me S-cHex S O O na 1,4-phenyl 14 HS-Et S-cHex S O O na 1,4-phenyl 15 Me S-Me S-(2R-EtOH) S O O na1,4-phenyl 16 Me S-Me S—iPr S N N H 1,4-phenyl 17 H S-Me S—iPr S N N H2,5- thiophenyl 18 Me S-Me S-cHex S N N H 2,5- thiophenyl 19 H S-MeS-cHex S N N H 1,4-phenyl 20 Me S-Me S-cHex S N N H 1,4-phenyl 21 MeS-Me S—iPr R N N H 1,4-phenyl 22 Me S-Me S-tBu R N N H 1,4-phenyl 23 MeS-Me S—iPr S N N H 1,4-phenyl 24 Me S-Me S-tBu R N N H 1,4-phenyl 25 MeS-Me S-(2R-EtOBn) S N N H 1,4-phenyl 26 Me S-Me S-(2R-EtOH) S N N H1,4-phenyl 27 Me S-Me S-(2R-EtOH) S N N H 1,4-phenyl 28 Me S-MeS-(2R-EtOH) R N N H 1,4-phenyl 29 Me S-Me S—iPr R N N H 1,4-phenyl 30 MeS-Me S-tBu R N N H 1,4-phenyl 31 Me S-Me S-(2R-EtOH) S N N H 1,4-phenyl32 Me S-Me S-(2R-EtOH) S N N H 1,4-phenyl 33 Me S-Me S-(2R-EtOBn) S N NH 1,4-phenyl 34 Me S-Me S-tBu R N N H 1,4-phenyl 35 Me S-Me S—iPr S N NH 1,4-phenyl 36 Me S-Me S-tBu R N N H 1,4-phenyl 37 Me S-Me S—iPr R N NH 1,4-phenyl 38 Me S-Me S-cHex S N N H 1,4-phenyl 39 H S-Me S-cHex S N NH 1,4-phenyl 40 Me S-Me S—iPr R N N H 1,4-phenyl 41 Me S-Me S-tBu R N NH 1,4-phenyl 42 H S-Me S-(2R-EtOH) S N N CH₂CH₂OH 1,4-phenyl 43 H S-MeS-(2R-EtOH) S N N CH₂CH₂OH 1,4-phenyl 44 Me S-Me S-(2R-EtOH) S N NCH₂CH₂OH 1,4-phenyl 45 H S-Me S-(2R-EtOH) S N N CH₂CH₂OAc 1,4-phenyl 46Me S-Me S—iPr S N N CH₂CH₂OH 1,4-phenyl 47 Me S-Me S—iPr S N N CH₂CH₂OAc1,4-phenyl 487 Me S-Me S-tBu S N N CH₂CH₂OH 1,4-phenyl 49 Me S-Me S-tBuS N N CH₂CH₂OAc 1,4-phenyl 50 Me S-Me S-(2R-EtOH) S N N CH₂CH₂OMe1,4-phenyl 51 Me S-Me S-tBu S N N CH₂CH₂OMe 1,4-phenyl 52 Me S-Me S—iPrS N N CH₂CH₂OMe 1,4-phenyl 53 H S-Me S—iPr S N N CH₂CH₂OMe 1,4-phenyl 54Me S-Me S-tBu S N N CH₂CH₂OH 1,4-phenyl 55 Me S-Me S—iPr S N N CH₂CH₂OH1,4-phenyl 56 Me S-Me S-(2R-EtOH) S N N CH₂CH₂OH 1,4-phenyl 57 Me S-MeS—iPr S N N Me 1,4-phenyl 58 Me S-Me S-(2R-EtOH) S N N Me 1,4-phenyl 59Me S-Me S-tBu S N N Me 1,4-phenyl 60 Me R-Me R-tBu R N N CH₂CH₂OH1,4-phenyl 61 H R-Me R-iPr R O O na 1,4-phenyl Entry R3a R3b R5b R7b R8bR12 R13 R14 K_(D) Range, 1 H H S—iPr S-Me H H H H A 2 H H S—iPr S-Me H HH H A 3 H H S—iPr S-Me H H H H A 4 H H S—iPr H H H H H A 6 H H S—iPrS-Me H H H H A 7 H H S-tBu S-Me H H H H A 8 H H S-tBu S-Me Me H H H A 9H H S-tBu S-Et H H H H B 10 H H S—iPr S-Me Me H H H A 11 H H S—iPr S-EtH H H H A 12 H H S-cHex S-Me H H H H A 13 H H S-cHex S-Me Me H H H A 14H H S-cHex S-Et H H H H B 15 H H S-(2R-EtOH) S-Me Me H H H A 16 H HS—iPr S-Me Me H F H A 17 H H S—iPr S-Me H H F H A 18 H H S-cHex S-Me MeH F H A 19 H H S-cHex S-Me H H F H A 20 H H S-cHex S-Me Me H F H A 21R-OH R-OH S—iPr S-Me Me H H H B 22 R-OH R-OH S-tBu S-Me Me H H H B 23 HH S—iPr S-Me Me F H H A 24 S—OH S—OH S-tBu S-Me Me H H H A 25 H HS-(2R-EtOBn) S-Me Me H F H A 26 H H S-(2R-EtOBn) S-Me Me H F H A 27 H HS-(2R-EtOH) S-Me Me H F H A 28 S—OH S—OH S-(2R-EtOH) S-Me Me H F H A 29S—OH S—OH S—iPr S-Me Me H F H A 30 S—OH S—OH S-tBu S-Me Me H F H A 31 HH S-(2R-EtOH) S-Me Me H F H A 32 H H S-(2R-EtOBn) S-Me Me H F H A 33 H HS-(2R-EtOBn) S-Me Me H F H A 34 S—OH S—OH S-tBu S-Me Me H H H A 35 H HS—iPr S-Me Me F H H A 36 R-OH R-OH S-tBu S-Me Me H H H B 37 R-OH R-OHS—iPr S-Me Me H H H B 38 H H S-cHex S-Me Me H F H A 39 H H S-cHex S-Me HH F H A 40 S—OH S—OH S—iPr S-Me Me F H H B 41 S—OH S—OH S-tBu S-Me Me FH H A 42 H H S-(2R-EtOBn) S-Me Me H F H A 43 H H S-(2R-EtOH) S-Me Me H FH A 44 H H S-(2R-EtOH) S-Me Me H F H A 45 H H S-(2R-EtOH) S-Me Me H F HA 46 H H S—iPr S-Me Me H F H A 47 H H S—iPr S-Me Me H F H A 487 H HS-tBu S-Me Me H F H A 49 H H S-tBu S-Me Me H F H A 50 H H S-(2R-EtOH)S-Me Me H F H A 51 H H S-tBu S-Me Me H F H A 52 H H S—iPr S-Me Me H F HA 53 H H S—iPr S-Me H H F H A 54 H H S-tBu S-Me Me F H H A 55 H H S—iPrS-Me Me F H H A 56 H H S-(2R-EtOH) S-Me Me F H H A 57 H H S—iPr S-Me MeH F H A 58 H H S-(2R-EtOH) S-Me Me H F H A 59 H H S-tBu S-Me Me H F H A60 H H R-tBu R-Me Me H F H C 61 H H R-iPr R-Me H H H H D

Further Examples

Example 2

Wherein R7a and R7b are independently H, alkyl, cycloalkyl, haloalkyl;or R8a and R7a and R8b and R7b can independently or together form a ringsuch as an aziridine or azetidine ring;

R8a and R8b are independently H, hydroxyl, alkyl, aryl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein eachalkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, andheteroarylalkyl is optionally-substituted with halogen, hydroxyl,mercapto, carboxyl, alkyl, alkoxy, amino, and nitro; or R8a and R7a andR8b and R7b can independently or together form a ring such as anaziridine or azetidine ring;

where R5a and R5b are independently H, alkyl, cycloalkyl,cycloalkylalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl; or each optionally-substituted with hydroxyl, mercapto,halogen, amino, carboxyl, alkyl, haloalkyl, alkoxy, or alkylthio; or, insome instances, the R5a and R5b residues are connected by an alkylene,alkenylene, alkynylene of 2 to 12 carbon atoms or optionally-substitutedalkylene, alkenylene, alkynylene bridge of 2 to 12 carbon atoms whereone or more carbon atoms can be replaced with N, O, or S;

R12a, R12b, R13a, R13b, R14a, and R14b are independently H, Cl, Br, F,alkyl, cycloalkyl, hydroxy, alkoxy, amino, alkylamino, cyano, or CO₂H;

R3a and R3b are independently H, halogen, alkyl, aryl, arylalkyl, amino,arylamino, arylalkylamino, hydroxy, alkyloxy, aryloxy, arylalkylhydroxy,dialkylamino, amido, sulfonamido, or amidino;

X and Y are independently O, N, S, or C═C;

R11a and R11b are independently absent, H, alkyl, optionally-substitutedalkyl, hydroxyalkyl, alkoxyalkyl; or R11a and R11b together form analkylene, alkenylene, alkynlyene, or alkyloxyalkylene chain of 2 to 12carbon atoms where one or more carbon atoms can be replaced with N, O,or S;

Wa and Wb together are a bond, alkylene, alkenylene, alkynylene, aryl,heteroaryl, or an optionally-substituted alkylene, alkenylene,alkynylene chain of 2 to 12 carbon atoms where one or more carbon atomscan be replaced with N, O, or S.

Stereochem at K_(D) Entry R8a R7a R5a Position (*) X Y R11 Wa-Wb R3a R3bR5b R7b R8b R12 R13 R14 Range, 62 H H S—iPr S O O na 1,4- H H S—iPr H HH H H B phenyl 62 H H S-tBu S N N CH₂CH₂OH 1,4- H H S-tBu H H H F H Bphenyl 63 H Me S-tBu S N N CH₂CH₂OAc 1,4- H H S-tBu Me H H F H B phenyl

Further Examples

Example 3

Wherein R7a and R7b are independently H, alkyl, cycloalkyl, haloalkyl;or R8a and R7a and R8b and R7b can independently or together form a ringsuch as an aziridine or azetidine ring;

R8a and R8b are independently H, hydroxyl, alkyl, aryl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein eachalkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, andheteroarylalkyl is optionally-substituted with halogen, hydroxyl,mercapto, carboxyl, alkyl, alkoxy, amino, and nitro; or R8a and R7a andR8b and R7b can independently or together form a ring such as anaziridine or azetidine ring;

R5a and R5b are independently H, alkyl, cycloalkyl, cycloalkylalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl; or eachoptionally-substituted with hydroxyl, mercapto, halogen, amino,carboxyl, alkyl, haloalkyl, alkoxy, or alkylthio; or, in some instances,the R5a and R5b residues are connected by an alkylene, alkenylene,alkynylene bridge of 2 to 12 carbon atoms or optionally-substitutedalkylene, alkenylene, alkynylene bridge of 2 to 12 carbon atoms whereone or more carbon atoms can be replaced with N, O, or S;

R12a, R12b, R13a, R13b, R14a, and R14b are independently H, Cl, Br, F,alkyl, cycloalkyl, hydroxy, alkoxy, amino, alkylamino, cyano, or CO₂H;

R3a and R3b are independently H, halogen, alkyl, aryl, arylalkyl, amino,arylamino, arylalkylamino, hydroxy, alkyloxy, aryloxy, arylalkylhydroxy,dialkylamino, amido, sulfonamido, or amidino;

X is O, N, S, or C═C;

R11a and R11b are independently absent, H, alkyl, optionally-substitutedalkyl, hydroxyalkyl, alkoxyalkyl; or R11a and R11b together form analkylene, alkenylene, alkynlyene, or alkyloxyalkylene chain of 2 to 12carbon atoms where one or more carbon atoms can be replaced with N, O,or S;

Wa and Wb together are a bond, alkylene, alkenylene, alkynylene, aryl,heteroaryl, or an optionally-substituted alkylene, alkenylene,alkynylene chain of 2 to 12 carbon atoms where one or more carbon atomscan be replaced with N, O, or S.

Stereochem at Position Entry R8a R7a R5a (*) X R11a Wa-Wb R3a R3b R5bR7b R8b R12 R13 R14 K_(D) Range 64 Me S-Me S—iPr S N CH₂CH₂OAc 1,4- H HS—iPr S-Me Me H F H A phenyl 65 H H S—iPr S N CH₂CH₂OAc 1,4- H H S—iPr HH H F H B phenyl 66 Me S-Me S-tBu S N CH₂CH₂OAc 1,4- H H S-tBu S-Me Me HF H A phenyl 67 Me S-Me S—iPr S N CH₂CH₂OH 1,4- H H S—iPr S-Me Me H F HA phenyl 68 H H S—iPr S N CH₂CH₂OH 1,4- H H S—iPr H H H F H C phenyl 69Me S-Me S-tBu S N CH₂CH₂OH 1,4- H H S-tBu S-Me Me H F H A phenyl 70 MeR-Me S—iPr S N CH₂CH₂OAc 1,4- H H S—iPr R-Me Me H F H A phenyl 71 MeR-Me S-tBu S N CH₂CH₂OH 1,4- H H S-tBu R-Me Me H F H B phenyl

See: Macor, J. E.; Blank, D. H.; Post, R. J.; Ryan, K. Tetrahedron Lett.1992, 33(52), 8011-8014.

2-(2-Ethoxycarbonyl-vinyl)-pyrrolidine-1-carboxylic acid tert-butylester (14)

A 2 L, 3-necked round bottomed flask equipped with an overhead stirredand a nitrogen inlet was charged with a solution of oxalyl chloride (130mL g, 0.26 mol) in DCM (250 mL). The solution was cooled to −78° C. Asolution of DMSO (20 mL, 0.28 mol) in DCM (30 mL) was added dropwise.After 30 min, a solution of alcohol 13 (40 g, 0.20 mol) in DCM (200 mL)was added dropwise. After 30 min, TEA (140 mL, 1.00 mol) was added tothe solution. The solution was transferred to an ice/water bath (0° C.)and stirring was continued for 30 min [NB: reaction mixture was a thick,white slurry]. TLC analysis revealed no remaining starting material [1:1hexane/EtOAc, R_(f)(13)=0.4; R_(f)(aldehyde)=0.6]. The reaction mixturewas diluted with DCM (200 mL) and washed successively with H₂O, 1 M HCl,saturated NaHCO₃, and brine. The DCM layer was dried over Na₂SO₄,filtered, and concentrated to afford crude2-formyl-pyrrolidine-1-carboxylic acid tert-butyl ester (40 g) as an oilwhich was used without further purification. ¹H NMR (CDCl₃, 300 MHz)δ9.50 (d, J=24 Hz, 1H), 4.20-4.03 (m, 1H), 3.60-3.40 (m, 2H), 2.20-1.87(m, 4H), 1.43 (s, 9H) ppm.

A 2 L, 3-necked round bottomed flask equipped with an overhead stirredand nitrogen inlet was charged with NaH (60%, 10.0 g, 0.25 mol) andanhydrous THF (200 mL). To the stirred mixture was slowly addedtriethylphosphonoacetate (53.8 g, 0.24 mol) over 20 minutes. A solutionof crude 2-formyl-pyrrolidine-1-carboxylic acid tert-butyl ester (40 g,0.20 mol) in THF (75 mL) was added dropwise. The solution turned orangeand the stirring was continued for 1 h until no aldehyde remained by TLCanalysis [1:1 hexane/EtOAc, R_(f)(aldehyde)=0.6; R_(f)(14)=0.8]. Thesolution was diluted with EtOAc and brine and the layers were separated.The EtOAc layer was washed with 1M HCl, brine, dried over anhydrousNa₂SO₄, filtered, and concentrated to afford 14 (67 g) as a yellow oilwhich was used without further purification. ¹H NMR (CDCl₃, 300 MHz)δ6.92-6.76 (m, 1H), 5.82 (d, 1H), 4.56-4.32 (m, 1H), 4.25-4.12 (m, 2H),3.48-3.27 (m, 2H), 2.20-1.98 (m, 1H), 1.91-1.72 (m, 2H), 1.43 (s, 9H),1.25 (t, 3H) ppm.

2-(3-Hydroxy-propenyl)-pyrrolidine-1-carboxylic acid tert-butyl ester(15)

A 2 L, 3-necked round bottomed flask equipped with an overhead stirredwas charged with 14 (67 g, 0.20 mol) and DCM (400 mL). The solution wascooled to −78° C. To this solution was slowly added boron trifluorideetherate (30 mL, 0.20 mol). The reaction mixture was stirred for 30 min.DIBAL (1 M in DCM, 600 mL, 0.6 mol) was added at a moderate rate. Thesolution was stirred at −78° C. for 2 h and then treated with EtOAc (100mL) over 30 min to discharge remaining reagent. The reaction mixture wasallowed to warm to −5° C. The reaction mixture was CAREFULLY quenched bythe dropwise addition of 1 M HCl. The reaction mixture was diluted withDCM and H₂O and made acidic to dissolve aluminum salts. The layers wereseparated and the organic phase was washed successively with diluteaqueous HCl, water, and brine. The DCM layer was dried over Na₂SO₄,filtered, and concentrated. The residue was purified by flashchromatography (SiO₂, 25% to 80% EtOAc/hexane) to afford 15 as a yellowoil (36 g, 79%). [TLC analysis, 1:1 hexane/EtOAc, R_(f)(14)=0.8;R_(f)(15)=0.2]. ¹H NMR (CDCl₃, 300 MHz) δ5.73-5.52 (m, 2H), 4.39-4.16(m, 1H), 4.15-4.04 (m, 2H), 3.46-3.25 (m, 2H), 2.92 (br s, 1H),2.08-1.93 (m, 1H), 1.92-1.79 (m, 2H), 1.78-1.62 (m, 1H), 1.42 (s, 9H)ppm.

trans-2S-(3-Methanesulfonyloxy-propenyl)-pyrrolidine-1-carboxylic acidtert-butyl ester (16)

To a solution of 15 (19 g, 84 mmol) in DCM (100 mL) was addedtriethylamine (10 g, 13.9 mL, 100 mmol). The solution was cooled to 0°C. and methanesulfonyl chloride (9.6 g, 6.5 mL, 84 mmol) in DCM (20 mL)was added dropwise. After 1 h, TLC analysis revealed completeconsumption of 15 [1:1 hexane/EtOAc, R_(f)(15)=0.2; R_(f)(16)=0.6].Brine was added and the product was extracted with DCM (3×50 mL). Theorganic extracts were combined and washed with 1 N HCl, water, brine,dried over anhydrous Na₂SO₄, filtered, and concentrated to afford 21.4 gof 16 which was used without purification. ¹H NMR (CDCl₃, 300 MHz)δ4.4-4.0 (m, 2H), 3.42-3.21 (m, 3H), 3.0 (s, 3H), 2.00-1.6 (m, 4H), 1.42(s, 9H) ppm.

2-{3-[Acetyl-(2-bromo-5-fluoro-phenyl)-amino]-propenyl}-pyrrolidine-1-carboxylicacid tert-butyl ester (18)

To a suspension of 60% NaH (9.2 g, 0.23 mol) in anhydrous DMF (150 mL)at 0° C. was added 2-bromo-5-fluoroacetanilide (17, 53.4 g, 0.23 mol) insmall portions. After 1 h, a solution of crude mesylate 16 (approx. 0.19mol) in DMF (20 mL) was added in a dropwise fashion from an additionfunnel. The reaction mixture was allowed to warm to ambient temperatureovernight. The reaction mixture was recooled to 0° C. and carefullyquenched by the addition of brine and neutralized by the addition ofdilute aqueous HCl until pH=7. The mixture was diluted with diethylether and water and the layers were separated. The organic phase waswashed several times with water to remove DMF followed by washing withbrine, dried over anhydrous Na₂SO₄, filtered, and concentrated. Thecrude product was purified by flash silica gel chromatography (0.5% to2% MeOH/DCM) to afford 66 g of 18 as an oil. [TLC analysis, 1:1hexane/EtOAc, R_(f)(16)=0.5; R_(f)(17)=0.6; R_(f)(18)=0.4]. ¹H NMR(CDCl₃, 300 MHz) δ7.64 (m, 1H), 7.01 (m, 2H), 5.52 (m, 1H), 5.39 (appdd, J=6.0, 15.3 Hz, 1H), 4.77 (app dd, J=4.5, 13.8 Hz, 1H), 4.24 (m,1H), 3.67 (app dd, J=7.5, 13.8 Hz, 1H), 3.32 (m, 2H), 1.90 (m, 1H), 1.81(m, 3H), 1.75 (m, 2H), 1.57 (m, 1H), 1.43 (m, 9H) ppm.

2-(1-Acetyl-6-fluoro-1H-indol-3-ylmethyl)-pyrrolidine-1-carboxylic acidtert-butyl ester (19)

Under a nitrogen atmosphere, a solution of 18 (66 g, 0.15 mol) inanhydrous DMF (350 mL) was charged with (n-Bu)₄NCl (41.5 g, 0.15 mol),K₂CO₃ (20.6 g, 0.15 mol), NaHCO₂ (10.2 g, 0.15 mol), and Pd(OAc)₂ (3.35g, 0.015 mol) at ambient temperature. The heterogeneous mixture wasimmersed in a pre-heated (85° C.) oil bath. After 1 h, TLC analysisrevealed some 18 remained therefore more catalyst (1 g) was added. After1.5 h, another charge of catalyst (0.6 g) was added. After an additional1.5 h of heating, 18 had been completely consumed by TLC analysis [TLCanalysis, 2% MeOH/DCM, R_(f)(18)=0.7; R_(f)(19)=0.8]. The warm reactionmixture was transferred to an ice water bath to cool then diluted withdiethyl ether and filtered through a pad of celite. The solids werewashed with diethyl ether and the filtrate was washed several times withwater to remove DMF then once with brine, dried over anhydrous Na₂SO₄,filtered, and concentrated to afford 52.5 g of crude 19, which was usedwithout further purification. ¹H NMR (CDCl₃, 300 MHz) δ8.18 (m, 1H),7.60 (m, 1H), 7.18 (m, 1H), 7.05 (dt, J=2.4, 8.7 Hz, 1H), 4.13 (m, 1H),3.41 (m, 1H), 3.33 (m, 2H), 3.17 (app dd, J=14.1, 38.1 Hz, 1H), 2.61 (s,3H), 1.83 (m, 3H), 1.69 (m, 1H), 1.49 (s, 9H) ppm.

2-(6-Fluoro-1H-indol-3-ylmethyl)-pyrrolidine-1-carboxylic acidtert-butyl ester (20)

A solution containing crude 19 (48 g) in reagent grade MeOH (480 mL) wascooled to 0° C. Aqueous NaOH (1 M, 144 mL) was added in one portion.After 30 min, TLC analysis revealed complete consumption of startingmaterial [TLC analysis, 3:2 hexane/EtOAc, R_(f)(19)=0.7; R_(f)(20)=0.8].The reaction mixture was neutralized with 1 N HCl and the product wasextracted with DCM. The DCM extracts were washed with water, brine,dried over anhydrous Na₂SO₄, filtered, and concentrated. The crudeproduct was absorbed onto 200 mL of silica gel and chromatographed (80%to 65% hexane/EtOAc) to afford 31.7 g of 20 as a thick oil. ¹H NMR(CDCl₃, 300 MHz) δ8.11 (br s, 1H), 7.65-7.57 (m, 1H), 7.04 (m, 1H), 6.96(s, 1H), 6.87 (t, J=2.8 Hz, 1H), 4.16-4.09 (m, 1H), 3.45-3.14 (m, 3H),2.76-2.63 (m, 1H), 1.75 (br s, 4H), 1.58 (s, 9H) ppm.

2-{1-[2-(tert-Butyl-dimethyl-silanyloxy)-ethyl]-6-fluoro-1H-indol-3-ylmethyl}-pyrrolidine-1-carboxylicacid tert-butyl ester (22)

Under a nitrogen atmosphere, a solution of 20 (3.0 g, 9.42 mmol) inanhydrous DMF (40 mL) was added via addition funnel to a mixture of 60%NaH (0.45 g, 11.3 mmol) in DMF (10 mL) at 0° C. After 1 h, bromide 21(2.47 g, 2.22 mL, 10.3 mmol) in DMF (5 mL) was added via syringe. After30 min, the reaction mixture was warmed to ambient temperature andstirred for an additional 30 min. The reaction was quenched by theaddition of saturated aqueous NH₄Cl and diluted with water. The productwas extracted with diethyl ether and the combined ether extracts werewashed several times with water to remove DMF, brine, dried overanhydrous Na₂SO₄, filtered, and concentrated to afford 4.49 g (quant.)of 22 as a yellow oil which was used without further purification. TLCanalysis [3:1 hexane/EtOAc, R_(f)(20)=0.4; R_(f)(22)=0.7]. ¹H NMR(CDCl₃, 300 MHz) δ7.68 (m, 1H), 7.12 (d, J=3.3 Hz, 1H), 7.03 (s, 1H),6.98 (t, J=3.2 Hz, 1H), 4.26-4.23 (m, 3H), 4.05-3.99 (m, 2H), 3.55-3.27(m, 3H), 2.75 (m, 1H), 1.88 (br s, 4H), 1.67 (s, 9H), 1.33 (m, 1H),1.06-1.00 (m, 3H), 0.95 (s, 9H), 0.23-0.14 (m, 2H) ppm.

2-[6-Fluoro-1-(2-hydroxy-ethyl)-1H-indol-3-ylmethyl]-pyrrolidine-1-carboxylicacid tert-butyl ester (23)

A solution containing 22 (4.49 g, 9.42 mmol) in anhydrous THF (50 mL)was cooled to 0° C. Tetra-n-butylammonium fluoride (1 M in THF, 14 mL,14 mmol) was added via syringe. After 1 h, reaction complete by TLCanalysis [3:1 hexane/EtOAc, R_(f)(22)=0.7; R_(f)(23)=0.1] thereforediluted with EtOAc. The EtOAc solution was washed twice with 1 M HCl,water, brine, dried over anhydrous Na₂SO₄, filtered, and concentrated toafford 3.9 g of 23 (>100%; contaminated with some TBS-containingimpurities) as a tan-colored oil which was used without furtherpurification. ¹H NMR (CDCl₃, 300 MHz) δ7.59 (br s, 1H), 7.01-6.85 (m,3H), 4.19-4.10 (m, 3H), 3.90 (br s, 2H), 3.38-3.31 (m, 2H), 3.15 (dd,J=1.4, 4.6 Hz, 1H), 2.68 (m, 1H), 1.79-1.72 (m, 4H), 1.47 (d, J=10.9 Hz,9H) ppm.

2-{6-Fluoro-1-[2-(toluene-4-sulfonyloxy)-ethyl]-1H-indol-3-ylmethyl}-pyrrolidine-1-carboxylicacid tert-butyl ester (12)

Triethylamine (1.13 g, 1.56 mL, 11.2 mmol) was added to a solution of 23(3.4 g, 9.38 mmol) in anhydrous DCM (50 mL) at 0° C. followed by theaddition of p-TsCl (1.79 g, 9.38 mmol) and DMAP (0.12 g, 0.94 mmol).After 30 min, the reaction mixture was warmed to room temperature. Uponcomplete consumption of 23 (˜30 min at ambient temperature), thereaction mixture was diluted with DCM and washed twice with 1 M HCl,brine, dried over anhydrous Na₂SO₄, filtered, and concentrated. Thecrude tosylate was purified by flash silica gel chromatography (3:1hexane/EtOAc) to afford 3.67 g (76%) of 24 as a white foam which washomogeneous by TLC analysis [3:1 hexane/EtOAc, R_(f)(23)=0.1;R_(f)(24)=0.3]. ¹H NMR (CDCl₃, 300 MHz) δ7.64-7.45 (m, 3H), 7.11 (t,J=2.5 Hz, 2H), 6.85 (dd, J=0.8, 3.3 Hz, 1H), 6.79 (s, 1H), 6.73 (t,J=3.6 Hz, 1H), 4.25 (s, 4H), 4.08 (br s, 1H), 3.34 (br d, J=9.6 Hz, 2H),3.20-3.09 (m, 1H), 2.64-2.57 (m, 1H), 2.36 (s, 1H), 1.75 (br s, 4H),1.53 (s, 9H) ppm.

1,2-Bis[2-(6-Fluoro-1H-indol-3-ylmethyl)-pyrrolidine-1-carboxylic acidtert-butyl ester]ethane (25)

To a suspension of 60% NaH (0.34 g, 8.50 mmol) in anhydrous DMF (20 mL)at 0° C. was added a solution of 20 (2.47 g, 7.75 mmol) in DMF (30 mL)via addition funnel. After 1 h, the reaction mixture was transferred toa −40° C. bath (ACN/dry ice). At −40° C., a solution of tosylate 24(3.65 g, 7.06 mmol) in DMF (20 mL) was added to the cold anion solutionfrom an addition funnel. After 30 min, only starting materials observedby TLC analysis therefore slowly warmed to 0° C. over 2 h. After 2-3 hat 0° C., the reaction was quenched by the addition of saturated aqueousNH₄Cl. The mixture was diluted with diethyl ether and water and thelayers were separated. The ether layer was washed several times withwater to remove DMF then once with brine, dried over anhydrous Na₂SO₄,filtered, and concentrated. The crude product was purified by normalphase HPLC (10-100% EtOAc/hexane over 30 min) to afford 3.27 g (70%) of25 as a white foam which was homogeneous by TLC analysis [3:1hexane/EtOAc (two developments), R_(f)(20)=0.8; R_(f)(24)=0.55;R_(f)(25)=0.5]. ¹H NMR (CDCl₃, 300 MHz) δ7.61-7.52 (m, 1H), 6.82 (t,J=9.6 Hz, 1H), 6.68-6.61 (m, 1H), 6.48-6.46 (m, 1H), 4.34 (s, 2H), 3.93(m, 1H), 3.34-3.26 (m, 2H), 3.17-3.01 (m, 1H), 2.05 (m, 1H), 1.70-1.58(m, 4H), 1.50 (s, 9H) ppm.

1,2-Bis[2-(6-Fluoro-1H-indol-3-ylmethyl)-pyrrolidine]ethane (26)

Trifluoroacetic acid (2 mL) was added at 0° C. to a solution containing25 (3.27 g, 4.93 mmol) in DCM (10 mL). After 3 h, an additional portionof TFA (2 mL) was added and the reaction was complete within 1 h. Thesolvent was removed on a rotary evaporator and the residue was dissolvedin DCM and washed twice with saturated aqueous NaHCO₃, once with brine,dried over anhydrous Na₂SO₄, filtered, and concentrated to afford 26 asa yellow foam which was used without further purification. ¹H NMR(CDCl₃, 300 MHz) δ7.31 (dd, J=5.1, 8.7 Hz, 1H), 6.92 (s, 1H), 6.77 (ddd,J=2.4, 9.6, 11.1 Hz, 1H), 6.44 (dd, J=2.4, 9.9 Hz, 1H), 4.41 (s, 2H),3.65-3.55 (m, 1H), 3.24-3.16 (m, 1H), 3.01-2.96 (m, 1H), 2.92 (d, J=7.8Hz, 2H), 2.15-1.99 (m, 1H), 1.96-1.84 (m, 2H), 1.76-1.67 (m, 1H) ppm.

1,2-Bis{2,2,2-Trifluoro-1-[2-(6-fluoro-1H-indol-3-ylmethyl)-pyrrolidin-1-yl]-ethanone}ethane(27)

At 0° C., TFAA (2.17 g, 1.44 mL, 10.3 mmol) was added to a solutioncontaining 26 (2.28 g, 4.93 mmol; based on theoretical yield fromprevious step) and TEA (2.49 g, 3.43 mL, 24.6 mmol) in DCM (50 mL).After 30 min, the reaction mixture is diluted with DCM and washed twicewith saturated aqueous NaHCO₃, once with brine, dried over anhydrousNa₂SO₄, filtered, and concentrated. The crude product was purified byflash silica gel chromatography (4:1 to 1:1 hexane/EtOAc) to afford 2.66g (82%, 2 steps) of 27 which was homogeneous by TLC analysis [2:1hexane/EtOAc, R_(f)(26)=0.01; R_(f)(27)=0.5]. ¹H NMR (CDCl₃, 300 MHz)δ7.70 (dd, J=5.4, 9.0 Hz, 1H), 6.84 (ddd, J=1.8, 9.3, 10.5 Hz, 1H), 6.62(dd, J=1.8, 10.2 Hz, 1H), 6.44 (s, 1H), 4.36 (s, 2H), 4.29-4.28 (m, 1H),3.60 (app t, J=7.2 Hz, 2H), 3.23 (dd, J=2.4, 14.1 Hz, 1H), 2.51 (dd,J=9.9, 14.1 Hz, 1H), 1.92-1.84 (m, 2H), 1.72-1.66 (m, 1H), 1.57-1.56 (m,1H) ppm.

1-(2-{3,10-Difluoro-14-[1-(2,2,2-trifluoro-acetyl)-pyrrolidin-2-ylmethyl]-6,7-dihydro-pyrazino[1,2-a;4,3-a′]diindol-13-ylmethyl}-pyrrolidin-1-yl)-2,2,2-trifluoro-ethanone(28)

Acyclic dimer 27 (2.66 g, 4.06 mmol) was dissolved in neat TFA (25 mL)at ambient temperature. After 3 h, the solvent was removed on a rotaryevaporator and the resultant residue was dissolved in EtOAc, washedtwice with saturated aqueous NaHCO₃, once with brine, dried overanhydrous Na₂SO₄, filtered, and concentrated to afford 2.65 g (quant.)of the diastereomeric indolylindolines as a yellow foam. [TLC analysis:3:1 hexane/EtOAc, R_(f)(27)=0.3; R_(f)(indolylindolines)=0.6-0.7].

To a mixture of crude indolylindolines (2.65 g, 4.05 mmol) in1,4-dioxane (50 mL) was added DDQ (1.10 g, 4.84 mmol) in one portion.After 2-3 h, the reaction mixture was diluted with EtOAc and filteredthrough a pad of celite. The solids were washed with EtOAc and thefiltrate was washed five times with saturated aqueous NaHCO₃, then oncewith brine. The combined aqueous washes were re-extracted twice withEtOAc and the combined organic extracts were dried over anhydrousNa₂SO₄, filtered, and concentrated. The crude product was purified byflash silica gel chromatography (4:1 hexane/EtOAc) to afford 1.94 g(73%, 2 steps) of 28 as an off-white solid which was homogeneous by TLCanalysis (2:1 hexane/EtOAc, R_(f)(indolylindolines)=0.6-0.7;R_(f)(28)=0.55]. NB: The product 2,2′-biindole (28) is quite fluorescentand is easily purified by trituration with reagent grade MeOH to afforda white solid. ¹H NMR (CDCl₃, 300 MHz) δ8.06 (dd, J=5.1, 8.1 Hz, 1H),7.03-6.93 (m, 2H), 4.49 (d, J=9.0 Hz, 1H), 4.40 (m, 1H), 4.12 (d, J=9.0Hz, 1H), 3.75-3.69 (m, 2H), 3.57-3.51 (m, 2H), 2.85 (dd, J=10.5, 12.9Hz, 1H), 1.78-1.74 (m, 2H), 1.51-1.45 (m, 1H) ppm.

3,10-Difluoro-13,14-bis-pyrrolidin-2-ylmethyl-6,7-dihydro-pyrazino[1,2-a;4,3-a′]diindole(29)

A mixture containing 28 (1.94 g, 2.97 mmol) and K₂CO₃ (2.05 g, 14.8mmol) in MeOH (60 mL) was heated at 60° C. for 1.5 h. The reactionmixture was cooled to ambient temperature and diluted with EtOAc andwater. The layers were separated and the aqueous phase was extractedthree times with EtOAc. The combined organic extracts were washed withbrine, dried over anhydrous Na₂SO₄, filtered, and concentrated to afford1.57 g (quant.) of 29 as a yellow solid which was used without furtherpurification. TLC analysis, 1:1 hexane/EtOAc, R_(f)(28)=0.9;R_(f)(29)=0.01. ¹H NMR (CDCl₃, 300 MHz) δ7.65 (m, 1H), 6.98 (app d,J=8.2 Hz, 1H), 6.90 (app t, J=8.3 Hz, 1H), 4.31 (s, 2H), 3.97 (br s,3H), 3.54 (m, 1H), 3.31 (m, 1H), 3.14 (m, 1H), 2.97 (m, 1H), 1.83 (m,1H), 1.68 (m, 2H), 1.42 (m, 1H) ppm. ¹³C NMR (CDCl₃, 75 MHz) δ160.6 (d,J_(C-F)=238.7 Hz), 136.2 (d, J_(C-F)=12.0 Hz), 127.1, 125.4, 120.8 (d,J_(C-F)=10.2 Hz), 109.8, 108.9 (d, J_(C-F)=24.6 Hz), 95.3 (d,J_(C-F)=26.3 Hz), 59.6, 45.6, 41.6, 31.0, 30.7, 24.5 ppm.

[1-(2-{14-[1-(2-tert-Butoxycarbonylamino-3-methyl-butyryl)-pyrrolidin-2-ylmethyl]-3,10-difluoro-6,7-dihydro-pyrazino[1,2-a;4,3-a′]diindol-13-ylmethyl}-pyrrolidine-1-carbonyl)-2-methyl-propyl]-carbamicacid tert-butyl ester (30)

A solution containing Boc-L-Val-OH (0.69 g, 3.18 mmol) and HATU (1.27 g,3.34 mmol) in anhydrous NMP (4 mL) was cooled to 0° C. After 15 min,DIPEA (0.45 g, 0.61 mL, 3.50 mmol) was added via syringe. After 15 min,a solution containing 29 (0.70 g, 1.52 mmol) in NMP (4 mL) was added andthe reaction mixture was allowed to warm to ambient temperature over 2 hat which point TLC analysis revealed complete consumption of 29 [TLCanalysis, 2:1 hexane/EtOAc, R_(f)(29)=0.01; R_(f)(30)=0.5]. The reactionmixture was diluted with diethyl ether and washed once with diluteaqueous HCl, five times with water to remove excess NMP, once withsaturated aqueous NaHCO₃ and brine, dried over anhydrous Na₂SO₄,filtered, and concentrated. The crude product was purified by flashsilica gel chromatography (3:1 hexane/EtOAc) to afford 1.09 g (83%) of30 as a pale yellow solid. ¹H NMR (CDCl₃, 300 MHz) δ8.04 (dd, J=5.1, 8.7Hz, 1H), 6.98 (m, 2H), 5.33 (d, J=9.3 Hz, 1H), 4.50 (m, 1H), 4.49 (d,J=8.1 Hz, 1H), 4.24 (dd, J=7.2, 9.3 Hz, 1H), 4.11 (m, 2H), 3.67 (dd,J=3.0, 13.5 Hz, 1H), 3.56 (m, 2H), 2.73 (app t, J=12.9 Hz, 1H), 1.99(dd, J=7.2, 13.5 Hz, 1H), 1.70-1.17 (m, 2H), 1.43 (s, 9H), 1.01 (d,J=7.2 Hz, 3H), 0.98 (d, J=7.5 Hz, 3H) ppm. ¹³C NMR (CDCl₃, 75 MHz)δ171.2, 160.4 (d, J_(C-F)=238 Hz), 155.7, 136.6 (d, J_(C-F)=12.0 Hz),127.2, 124.8, 122.0 (d, J_(C-F)=9.7 Hz), 109.2, 108.5 (d, J_(C-F)=24.0Hz), 95.0 (d, J_(C-F)=26.3 Hz), 79.4, 57.7, 56.9, 47.3, 41.7, 31.8,29.7, 28.4, 28.3, 23.8, 19.7, 17.7 ppm.

2-Amino-1-(2-{14-[1-(2-amino-3-methyl-butyryl)-pyrrolidin-2-ylmethyl]-3,10-difluoro-6,7-dihydro-pyrazino[1,2-a;4,3-a]diindol-13-ylmethyl}-pyrrolidin-1-yl)-3-methyl-butan-1-one(31)

A solution containing 30 (1.09 g, 1.27 mmol) in DCM (20 mL) was cooledto 0° C. TFA (4 mL) was added via pipette and the reaction was monitoreduntil TLC analysis revealed complete consumption of 30 (˜2 h). TLCanalysis, 10% MeOH/DCM, R_(f)(30)=0.5; R_(f)(31)=0.4. The solvent wasremoved on a rotary evaporator and the residue was dissolved in EtOAc.The EtOAc solution was washed twice with saturated aqueous NaHCO₃ andonce with brine. The combined aqueous washes were back-extracted withEtOAc and the organic extracts were dried over anhydrous Na₂SO₄,filtered, and concentrated to afford 0.83 g (quant.) of 31 as a yellowsolid which was used without further purification. ¹H NMR (CDCl₃, 300MHz) δ8.09 (dd, J=5.1, 8.7 Hz, 1H), 6.97 (m, 2H), 4.52 (m, 1H), 4.50 (d,J=8.7 Hz, 1H), 4.11 (m, 1H), 3.71 (br d, J=11.1 Hz, 1H), 3.51-3.32 (m,2H), 2.74 (app t, J=12.6 Hz, 1H), 2.30 (br s, 4H), 1.92 (m, 1H), 1.68(m, 2H), 1.41 (m, 1H), 1.03 (m, 6H) ppm. ¹³C NMR (CDCl₃, 75 MHz) δ174.3,171.4, 160.6 (d, J_(C-F)=232.5 Hz), 136.8 (d, J_(C-F)=7.5 Hz), 127.4 (d,J_(C-F)=3.7 Hz), 125.0, 122.4 (d, J_(C-F)=7.5 Hz), 109.6, 108.7 (d,J_(C-F)=22.5 Hz), 95.2 (d, J_(C-F)=22.5 Hz), 58.0, 47.3, 41.9, 30.0,28.5, 28.5, 24.1, 19.9, 17.6 ppm.

Penultimate Intermediate (32):

A solution containing Boc-L-N(Me)Ala-OH (0.49 g, 2.45 mmol) and HATU(0.98 g, 2.56 mmol) in anhydrous NMP (4 mL) was cooled to 0° C. After 15min, DIPEA (0.35 g, 0.47 mL, 2.69 mmol) was added via syringe. After 15min, a solution containing 31 (0.77 g, 1.17 mmol) in NMP (4 mL) wasadded and the reaction mixture was allowed to warm to ambienttemperature over 2 h at which point TLC analysis revealed completeconsumption of 31 [TLC analysis, 1:1 hexane/EtOAc, R_(f)(31)=0.01;R_(f)(32)=0.5]. The reaction mixture was diluted with diethyl ether andwashed once with dilute aqueous HCl, five times with water to removeexcess NMP, once with saturated aqueous NaHCO₃ and brine, dried overanhydrous Na₂SO₄, filtered, and concentrated. The crude product waspurified by flash silica gel chromatography (1:1 hexane/EtOAc) to afford0.92 g (76%) of 32 as a pale yellow solid. ¹H NMR (CDCl₃, 300 MHz) δ8.05(m, 1H), 7.65-6.90 (m, 2H), 4.53 (m, 3H), 4.13 (m, 1H), 3.70-3.52 (m,4H), 2.82 (m, 2H), 2.72 (app t, J=11.1 Hz, 1H), 1.70 (m, 1H), 1.64 (s,3H), 1.53 (s, 9H), 1.45-1.25 (m, 2H), 1.34 (d, J=7.0 Hz, 3H), 1.05-0.88(m, 6H) ppm. ¹³C NMR (CDCl₃, 75 MHz) δ171.3, 170.4, 160.4 (d,J_(C-F)=232.5 Hz), 136.6 (d, J_(C-F)=7.5 Hz), 127.2, 124.7, 122.1,109.2, 108.5 (d, J_(C-F)=22.5 Hz), 95.0 (d, J_(C-F)=22.5 Hz), 57.8,55.5, 47.4, 41.7, 31.6, 29.9, 29.7, 28.4, 23.8, 19.3, 18.0 ppm.

N-{1-[2-(3,10-Difluoro-14-{1-[3-methyl-2-(2-methylamino-propionylamino)-butyryl]-pyrolidin-2-ylmethyl}-6,7-dihydro-pyrazino[1,2-a;4,3-a′]diindol-13-ylmethyl)-pyrrolidine-1-carbonyl]-2-methyl-propyl}-2-methylamino-propionamide(33)

A solution containing 32 (0.92 g, 0.89 mmol) in DCM (15 mL) was cooledto 0° C. TFA (3 mL) was added via pipette and the reaction was monitoreduntil TLC analysis revealed complete consumption of 32 (˜3 h). TLCanalysis, 10% MeOH/DCM, R_(f)(32)=0.4; R_(f)(33)=0.3. The solvent wasremoved on a rotary evaporator and the residue was dissolved in EtOAc.The EtOAc solution was washed twice with saturated aqueous NaHCO₃ andonce with brine. The combined aqueous washes were back-extracted withEtOAc and the organic extracts were dried over anhydrous Na₂SO₄,filtered, and concentrated to afford 0.73 g of crude 33. The crudeproduct was purified by RP-HPLC (Method: Solvent A: water w/0.1% v/vHOAc, Solvent B: ACN w/0.1% v/v HOAc. Dynamax Microsorb C18 60 Å 8μ,41.4 mm×25 cm; Flow: 40 mL/min; Detector: 272 nm). Theproduct-containing fractions were diluted with saturated aqueous NaHCO₃and extracted with EtOAc. The EtOAc extract was washed with brine, driedover anhydrous Na₂SO₄, filtered, and concentrated. The residue wasdissolved in a minimum amount of ACN, diluted with water until cloudy,frozen, and lyophilized to afford 33 as a flocculent, white solid. ¹HNMR (DMSO, 300 MHz) δ8.04-7.86 (m, 2H), 7.38 (app dd, J=2.3, 10.5 Hz,1H), 6.90 (app dt, J=2.3, 9.9 Hz, 1H), 4.68 (app d, J=8.7 Hz, 1H),4.34-4.23 (m, 2H), 3.98 (d, J=8.7 Hz, 1H), 3.46 (m, 2H), 2.94 (app q,J=6.4 Hz, 1H), 2.70 (t, J=12.8 Hz, 1H), 2.12 (s, 3H), 1.94 (m, 1H), 1.58(m, 2H), 1.35 (m, 1H), 1.16-1.07 (m, 2H), 1.03 (d, J=7.0 Hz, 3H), 0.85(m, 6H) ppm. ¹³C NMR (CDCl₃, 75 MHz) δ175.0, 170.8, 160.6 (d,J_(C-F)=232.5 Hz), 136.8 (d, J_(C-F)=7.5 Hz), 127.4 (d, J_(C-F)=3.7 Hz),125.0, 122.3 (d, J_(C-F)=7.5 Hz), 109.5, 108.7 (d, J_(C-F)=22.5 Hz),95.2 (d, J_(C-F)=22.5 Hz), 60.4, 57.9, 55.3, 47.6, 42.0, 35.2, 31.7,30.0, 28.6, 24.0, 19.7, 19.6, 18.2 ppm. Mass spectrum, m/z=[415.6](M+2)+/2.

EXAMPLES

Example 4

Wherein R7a and R7b are independently H, alkyl, cycloalkyl, haloalkyl;or R8a and R7a and R8b and R7b can independently or together form a ringsuch as an aziridine or azetidine ring;

R8a and R8b are independently H, hydroxyl, alkyl, aryl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein eachalkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, andheteroarylalkyl is optionally-substituted with halogen, hydroxyl,mercapto, carboxyl, alkyl, alkoxy, amino, and nitro; or R8a and R7a andR8b and R7b can Independently or together form a ring such as anaziridine or azetidine ring;

R5a and R5b are independently H, alkyl, cycloalkyl, cycloalkylalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl; or eachoptionally-substituted with hydroxyl, mercapto, halogen, amino,carboxyl, alkyl, haloalkyl, alkoxy, or alkylthio; or, optionally, R5aand R5b are connected by an alkylene, alkenylene, alkynylene of 2 to 12carbon atoms or optionally-substituted alkylene, alkenylene, alkynylenebridge of 2 to 12 carbon atoms where one or more carbon atoms can bereplaced with N, O, or S;

R12a, R12b, R13a, R13b, R14a, and R14b are independently H, Cl, Br, F,alkyl, cycloalkyl, hydroxy, alkoxy, amino, alkylamino, cyano, or CO₂H;and

R3a and R3b are independently H, halogen, alkyl, aryl, arylalkyl, amino,arylamino, arylalkylamino, hydroxy, alkyloxy, aryloxy, arylalkylhydroxy,dialkylamino, amido, sulfonamido, or amidino.

Stereochem at Position Entry R8a R7a R5a (*) R3a R3b R5b R7b R8b R12 R13R14 K_(D) Range, 72 Me S-Me S-(2R-EtOH) S H H S-(2R-EtOH) S-Me Me H F HA 73 Me S-Me S—iPr S H H S—iPr S-Me Me H F H A 74 H S-Me S—iPr S H HS—iPr S-Me Me H F H A 75 Me S-Me S—iPr S H H S—iPr S-Me Me F H H A 76 MeS-Me H S H H H S-Me Me H F H B 77 Me S-Me S-Me S H H S-Me S-Me Me H F HB 78 Me S-Me S-(2S-EtOH) S H H S-(2S-EtOH) S-Me Me H F H A 79 Me S-MeS-Et S H H S-Et S-Me Me H F H A 80 Me S-Me S-(2S-EtOH) S H H S-(2S-EtOH)S-Me Me F H H A 81 H H S—iPr S H H S—iPr H H H F H B 82 Me S-Me S-sBu SH H S-sBu S-Me Me F H H A 83 Me S-Me S-cHex S H H S-cHex S-Me Me F H H A84 Me S-Me S-tBu S H H S-tBu S-Me Me H F H A 85 Me S-Me S-cHex S H HS-cHex S-Me Me H F H A 86 Me S-Me S-(2R-EtOH) R S—OH S—OH S-(2R-EtOH)S-Me Me H F H A 87 Me S-Me S—iPr R S—OH S—OH S—iPr S-Me Me H F H A 88 MeS-Me S-(2R-EtOMe) R S—OH S—OH S-(2R-EtOMe) S-Me Me H F H A 89 Me S-MeS-(2R-EtOtBu) R S—OH S—OH S-(2R-EtOtBu) S-Me Me H F H B 90 Me S-MeS-(2S-EtOH) R S—OH S—OH S-(2S-EtOH) S-Me Me H F H A 91 Me R-Me S—iPr S HH S—iPr R-Me Me H F H B 92 Me S-Me R-iPr S H H R-iPr S-Me Me H F H C 93Me R-Me R-iPr S H H R-iPr R-Me Me H F H C

Further Examples

Example 5

Wherein R7a and R7b are independently H, alkyl, cycloalkyl, haloalkyl;or R8a and R7a and R8b and R7b can independently or together form a ringsuch as an aziridine or azetidine ring;

R8a and R8b are independently H, hydroxyl, alkyl, aryl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein eachalkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, andheteroarylalkyl is optionally-substituted with halogen, hydroxyl,mercapto, carboxyl, alkyl, alkoxy, amino, and nitro; or R8a and R7a andR8b and R7b can independently or together form a ring such as anaziridine or azetidine ring;

R5a and R5b are independently H, alkyl, cycloalkyl, cycloalkylalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl; or eachoptionally-substituted with hydroxyl, mercapto, halogen, amino,carboxyl, alkyl, haloalkyl, alkoxy, or alkylthio; or, optionally, R5aand R5b are connected by an alkylene, alkenylene, alkynylene of 2 to 12carbon atoms or optionally-substituted alkylene, alkenylene, alkynylenebridge of 2 to 12 carbon atoms where one or more carbon atoms can bereplaced with N, O, or S;

R12a, R12b, R13a, R13b, R14a, and R14b are independently H, Cl, Br, F,alkyl, cycloalkyl, hydroxy, alkoxy, amino, alkylamino, cyano, or CO₂H;and

R3a and R3b are independently H, halogen, alkyl, aryl, arylalkyl, amino,arylamino, arylalkylamino, hydroxy, alkyloxy, aryloxy, arylalkylhydroxy,dialkylamino, amido, sulfonamido, or amidino.

Stereochem at Position K_(D) Entry R8a R7a R5a (*) R3a R3b R5b R7b R8bR12 R13 R14 Range 94 Me S-Me S-(2R-EtOH) S H H S-(2R-EtOH) S-Me Me H H HA

Bis-(Boc-Allylglycine)-Containing Species (34)

A solution containing Boc-L-allyl-Gly-OH (0.115 g, 0.53 mmol) and HATU(0.20 g, 0.53 mmol) in anhydrous NMP (3 mL) was cooled to 0° C. After 10min, diisopropylethylamine (0.1 mL, 0.58 mmol) was added via syringe.After 5 min, a solution containing 29 (0.11 g, 0.23 mmol) in NMP (3 mL)was added and the reaction mixture was allowed to warm to ambienttemperature over 16 h at which point TLC analysis revealed completeconsumption of 29 [TLC analysis, 5% MeOH/DCM, R_(f)(29)=0.4]. Thereaction mixture was diluted with diethyl ether and washed once withsaturated aqueous NaHCO₃, once with dilute aqueous HCl, and twice withbrine, dried over anhydrous Na₂SO₄, filtered, and concentrated. Thecrude product was purified by RP-HPLC (Dynamax Microsorb C18 60 Å 8μ,41.4 mm×250 mm; Flow 40 mL/min; Detector: 254 nm, 20-100% gradient ofACN/water with 0.1% AcOH over 30 min). The product-containing fractionswere diluted with EtOAc, washed with saturated aqueous NaHCO₃, driedover anhydrous Na₂SO₄, filtered, and concentrated to afford 0.067 g(73%) of 34 as a light yellow solid. ¹H NMR (CDCl₃, 300 MHz) δ8.06 (dd,J=5.4, 8.7 Hz, 1H), 7.02-6.92 (m, 2H), 5.88-5.79 (m, 1H), 5.42 (d, J=8.4Hz, 1H), 5.19-5.11 (m, 2H), 4.49 (dd, J=6.9, 15.9 Hz, 2H), 4.16-4.08 (m,1H), 3.65-3.41 (m, 3H), 2.75 (app t, J=12.9 Hz, 1H), 2.54-2.35 (m, 2H),1.75-1.68 (m, 3H), 1.46-1.43 (m, 9H), 1.21-1.19 (m, 1H) ppm. Massspectrum, m/z=877.7 (M+Na)+.

Ring Closing Metathesis (RCM) Product (35)

To a solution of 34 (0.067 g, 0.08 mmol) in anhydrous DCM (30 mL) wasadded the first generation Grubbs' catalyst (9.2 mg, 0.01 mmol, 12 mol%) at room temperature. The reaction mixture was heated under reflux for6 h at which time TLC analysis revealed mostly starting material.Additional Grubbs' catalyst (7 mg, 0.009 mmol, 11 mol %) was then addedto the reaction mixture. After 2 d, the solvent was evaporated and thecrude residue was purified by NP—HPLC (SiO₂, 20% hexane/EtOAc to 100%EtOAc over 20 min) to afford the desired olefins 35 (olefin isomers: 15mg and 24 mg) as a separable mixture of isomers (unassigned olefingeometry) as light yellow solids. [TLC analysis, 1:1 hexane/EtOAc,R_(f)(34)=0.7; R_(f)(35)=0.6]. Isomer A: ¹H NMR (CDCl₃, 300 MHz) δ7.80(app t, J=9.0 Hz, 1H), 7.00 (dd, J=1.5, 11.07 Hz, 1H), 6.92 (ddd, J=2.1,9.3, 11.2 Hz, 1H), 5.62 (app d, J=7.8 Hz, 1H), 5.56 (br s, 1H), 4.79 (m,1H), 4.56-4.47 (m, 2H), 4.12 (app d, J=6.9 Hz, 1H), 3.85 (dd, J=3.6,13.5 Hz, 1H), 3.52-3.49 (m, 1H), 3.41 (m, 1H), 2.73 (m, 1H), 2.45-2.40(m, 2H), 1.70-1.64 (m, 4H), 1.44 (s, 9H), 1.07-1.05 (m, 1H) ppm. Massspectrum, m/z=849.7 (M+Na)+. 35 Isomer B: ¹H NMR (CDCl₃, 300 MHz) δ7.85(br s, 1H), 7.0 (dd, J=1.5, 9.3 Hz, 1H), 6.92 (ddd, J=2.4, 9.3, 11.1 Hz,1H), 5.66 (br s, 1H), 5.56 (app d, J=7.5 Hz, 1H), 4.80 (br s, 1H),4.62-4.51 (m, 2H), 4.17-4.09 (m, 1H), 3.75-3.52 (m, 3H), 2.69-2.60 (m,2H), 2.47 (app d, J=15.3 Hz, 1H), 1.69 (m, 3H), 1.45 (s, 9H), 1.18 (m,1H) ppm. Mass spectrum, m/z=849.7 (M+Na)⁺.

Alkyl-Linked Product (36)

To a solution of olefin 35 Isomer A (15 mg, 0.02 mmol) in EtOAc (5 mL)was added 5% Pd/C (25 mg). The reaction mixture was shaken under a H₂atmosphere using a Parr apparatus (˜45-50 PSI). After 2.5 h, TLCanalysis revealed unreacted starting material. Additional 5% Pd/C (20mg) was then added and the mixture was again subjected to hydrogenationusing a Parr apparatus. After 1.5 h, the mixture was filtered throughCelite®, and the solids were rinsed with EtOAc. The filtrate wasconcentrated in vacuo to give 36 as a light yellow solid.

35 Isomer B (24 mg, 0.03 mmol) was subjected to the same reactionconditions and workup procedures as described for Isomer A. The product(36) was combined with that from the hydrogenation of Isomer A. Compound36 was isolated as a light yellow solid (35 mg, 85%). [TLC analysis, 1:1hexane/EtOAc, R_(f)(36)=0.3]. ¹H NMR (CDCl₃, 300 MHz) δ7.85 (dd, J=5.4,8.1 Hz, 1H), 7.00 (dd, J=1.8, 9.9 Hz, 1H), 6.92 (ddd, J=2.4, 9.2, 11.1,1H), 5.70 (d, J=7.8 Hz, 1H), 4.79-4.78 (m, 1H), 4.57-4.51 (m, 2H),4.13-4.09 (m, 1H), 3.75 (dd, J=3.6, 12.9 Hz, 1H), 3.53-3.41 (m, 2H),2.49 (app t, J=12.3 Hz, 1H), 1.68-1.62 (m, 3H), 1.46 (s, 9H), 1.08-1.02(m, 1H), 0.93-0.89 (m, 1H) ppm. Mass spectrum, m/z=851.7 (M+Na)+.

Free Alkyl-Linked Diamine (37)

A solution containing 36 (0.035 g, 0.04 mmol) in DCM (10 mL) was cooledto 0° C. TFA (1 mL) was added via pipette and the reaction was allowedto warm to ambient temperature and monitored until TLC analysis revealedcomplete consumption of 36 (˜1 h). The solvent was removed on a rotaryevaporator and the residue was dissolved in EtOAc. The EtOAc solutionwas washed twice with saturated aqueous NaHCO₃ and once with brine. Thecombined aqueous washes were back-extracted with EtOAc and the organicextracts were dried over anhydrous Na₂SO₄, filtered, and concentrated toafford 0.025 g (quant.) of 37 as a yellow solid which was used withoutfurther purification. ¹H NMR (CDCl₃, 300 MHz) δ7.92-7.87 (m, 1H), 7.00(dd, J=2.1, 9.9 Hz, 1H), 6.91 (ddd, J=2.4, 9.3, 11.1 Hz, 1H), 4.81 (m,1H), 4.52 (d, J=8.1 Hz, 1H), 4.13-4.09 (m, 1H), 3.79-3.76 (m, 2H),3.42-3.39 (m, 2H), 2.50 (app t, J=12.9 Hz, 1H), 1.89 (m, 4H), 1.65-1.61(m, 3H), 1.33-1.24 (m, 4H), 1.09-1.03 (m, 1H), 0.94-0.82 (m, 1H) ppm.Mass spectrum, m/z=315.3 (M+H)+/12; m/z=629.5 (M+H)+.

Bis-[Boc-N(Me)-Alanine]-Containing Macrocycle (38)

A solution containing Boc-N-methyl-L-Ala-OH (0.022 g, 0.11 mmol) andHATU (0.03 g, 0.09 mmol) in anhydrous NMP (4 mL) was cooled to 0° C.After 10 min, diisopropylethylamine (0.05 mL, 0.29 mmol) was added viasyringe. After 5 min, a solution containing 37 (0.025 g, 0.04 mmol) inNMP (3 mL) was added and the reaction mixture was allowed to warm toambient temperature over 24 h at which point TLC analysis revealedcomplete consumption of 37 [TLC analysis, 5% MeOH/DCM, R_(f)(38)=0.3].The reaction mixture was diluted with diethyl ether and washed once withsaturated aqueous NaHCO₃, once with dilute aqueous HCl, and twice withbrine, dried over anhydrous Na₂SO₄, filtered, and concentrated to give38 as a yellow oil (35 mg) which was used without further purification.

Macrocyclic Smac Mimetic (39)

A solution containing 38 (0.035 g, 0.04 mmol) in DCM (10 mL) was cooledto 0° C. TFA (1 mL) was added via pipette and the reaction was allowedto warm to ambient temperature and monitored until TLC analysis revealedcomplete consumption of 38 (˜2 h). The solvent was removed on a rotaryevaporator and the residue was dissolved in EtOAc. The EtOAc solutionwas washed twice with saturated aqueous NaHCO₃ and once with brine. Thecombined aqueous washes were back-extracted with EtOAc and the organicextracts were dried over anhydrous Na₂SO₄, filtered, and concentrated toafford 39 as a yellow solid. The crude product was purified by RP-HPLC(Dynamax Microsorb C18 60 Å 8μ, 41.4 mm×250 mm; Flow 40 mL/min; Detector254 nm, 20-100% gradient of ACN/water with 0.1% AcOH over 30 min). Theproduct-containing fractions were diluted with EtOAc and washed withsaturated aqueous NaHCO₃, dried over anhydrous Na₂SO₄, filtered, andconcentrated. The material was dissolved in a minimum amount of ACN,diluted with water, frozen, and lyophilized to afford 39 as a whiteflocculent solid (0.002 g). ¹H NMR (CDCl₃, 300 MHz) δ8.16 (app d, J=8.1Hz, 1H), 7.90-7.86 (m, 1H), 7.00 (app d, J=8.7 Hz, 1H), 6.93 (ddd,J=3.0, 7.5, 9.0 Hz, 1H), 4.80 (m, 2H), 4.52 (d, J=8.4 Hz, 1H), 4.11 (d,J=8.4 Hz, 1H), 3.79 (dd, J=3.9, 12.9 Hz, 1H), 3.51 (m, 1H), 3.44 (m,1H), 3.11-3.09 (m, 1H), 2.46 (s, 3H), 1.35-1.25 (m, 8H), 1.06 (m, 1H),0.89-0.85 (m, 1H) ppm. Mass spectrum, m/z=400.5 (M)+/2; m/z=799.7 (M)⁺;m/z=821.7 (M+Na+.

EXAMPLES

Example 6

Wherein R7a and R7b are independently H, alkyl, cycloalkyl, haloalkyl;or R8a and R7a and R8b and R7b can independently or together form a ringsuch as an aziridine or azetidine ring;

R8a and R8b are independently H, hydroxyl, alkyl, aryl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein eachalkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, andheteroarylalkyl is optionally-substituted with halogen, hydroxyl,mercapto, carboxyl, alkyl, alkoxy, amino, and nitro; or R8a and R7a andR8b and R7b can independently or together form a ring such as anaziridine or azetidine ring;

Z is a bond; an alkylene, alkenylene, alkynylene group of 1 to 6 atoms;or, an optionally substituted alkylene, alkenylene, or alkynylene groupof 1 to 6 carbon atoms; a sulfide (—S—), sulfoxide (—SO—), sulfone(—SO₂—), or disulfide (—SS—) group; an aryl, arylalkylene, heteroaryl,heteroarylalkylene, or an optionally substituted aryl, arylalkylene,heteroaryl, heteroarylalkylene group; an amino or substituted aminogroup; an oxygen atom;

m and n are independently 0, 1, 2, or 3;

R12a, R12b, R13a, R13b, R14a, and R14b are independently H, Cl, Br, F,alkyl, cycloalkyl, hydroxyl, alkoxy, amino, alkylamino, cyano, or CO₂H;and

R3a and R3b are independently H, halogen, alkyl, aryl, arylalkyl, amino,arylamino, arylalkylamino, hydroxy, alkyloxy, aryloxy, arylalkylhydroxy,dialkylamino, amido, sulfonamido, or amidino.

K_(D) Range, Entry R8a R7a m n Z R3a R3b R7b R8b R12 R13 R14 μM 95 Me Me1 1 Z-CH═CH— H H Me Me H F H A 96 Me Me 1 1 E-CH═CH— H H Me Me H F H A97 Me Me 1 1 —CH₂CH₂— H H Me Me F H H A 98 Me Me 1 1 —CH₂CH₂— H H Me MeH F H A 99 H Me 1 1 —SS— H H Me H H F H A 100 Me Me 1 1 —CH(OH)CH(OH)— HH Me Me H H H A 101 Me Me 1 1 —CH₂CH₂— S—OH S—OH Me Me H F H A

Further Examples

Example 7

Wherein R7a and R7b are independently H, alkyl, cycloalkyl, haloalkyl;or R8a and R7a and R8b and R7b can independently or together form a ringsuch as an aziridine or azetidine ring;

R8a and R8b are independently H, hydroxyl, alkyl, aryl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein eachalkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, andheteroarylalkyl is optionally-substituted with halogen, hydroxyl,mercapto, carboxyl, alkyl, alkoxy, amino, and nitro; or R8a and R7a andR8b and R7b can independently or together form a ring such as anaziridine or azetidine ring;

R5a and R5b are independently H, alkyl, cycloalkyl, cycloalkylalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl; or eachoptionally-substituted with hydroxyl, mercapto, halogen, amino,carboxyl, alkyl, haloalkyl, alkoxy, or alkylthio; or, optionally, R5aand R5b are connected by an alkylene, alkenylene, alkynylene of 2 to 12carbon atoms or optionally-substituted alkylene, alkenylene, alkynylenebridge of 2 to 12 carbon atoms where one or more carbon atoms can bereplaced with N, O, or S;

R12a, R12b, R13a, R13b, R14a, and R14b are independently H, Cl, Br, F,alkyl, cycloalkyl, hydroxy, alkoxy, amino, alkylamino, cyano, or CO₂H;and

R3a and R3b are independently H, halogen, alkyl, aryl, arylalkyl, amino,arylamino, arylalkylamino, hydroxy, alkyloxy, aryloxy, arylalkylhydroxy,dialkylamino, amido, sulfonamido, or amidino.

Stereochem at Position K_(D) Entry R8a R7a R5a (*) R3a R3b R5b R7b R8bR12 R13 R14 Range 102 H S-Me S—iPr S H H S—iPr S-Me Me H F H A 103 MeS-Me S-(2R-EtOH) S H H S-(2R-EtOH) S-Me Me H F H A 104 H S-Me S—iPr S HH S—iPr S-Me Me H H H A 105 Me S-Me S-(2R-EtOH) S H H S-(2R-EtOH) S-MeMe H H H A 106 H S-Me S-(2R-EtOH) S H H S-(2R-EtOH) S-Me H H H H A 107Me S-Me S—iPr S H H S—iPr S-Me Me H H H A 108 H S-Et S—iPr S H H S—iPrS-Et H H H H A 109 Me S-Et S—iPr S H H S—iPr S-Et Me H H H A 110 Me S-MeS-(2S-EtOH) S H H S-(2S-EtOH) S-Me Me H F H A 111 Me S-Me S-Allyl S H HS-Allyl S-Me Me H F H A 112 Me S-Me S—iPr R S—OH S—OH S—iPr S-Me Me H FH A 113 Me S-Me S-sBu R S—OH S—OH S—sBu S-Me Me H F H A 114 Me S-MeS-(2S-EtOtBu) R S—OH S—OH S-(2S-EtOtBu) S-Me Me H F H A 115 Me S-MeS-(2S-EtOH) R S—OH S—OH S-(2S-EtOH) S-Me Me H F H A 116 Me S-MeS-(2R-EtOMe) R S—OH S—OH S-(2R-EtOMe) S-Me Me H F H A 117 Me S-MeS-(2S-EtOMe) R S—OH S—OH S-(2S-EtOMe) S-Me Me H F H A 118 Me S-MeS-(2R-EtOH) R S—OH S—OH S-(2R-EtOH) S-Me Me H F H A

Preparation of2-Amino-N-[1-(2-{1-[6-(3-{1-[2-(2-amino-propionylamino)-3-methyl-butyryl]-pyrrolidin-2-ylmethyl}-indol-1-yl)-hexa-2,4-diynyl]-1H-indol-3-ylmethyl}-pyrrolidine-1-carbonyl)-2-methyl-propyl]-propionamide(43) A.(1S-{2-Methyl-1S-[2S-(1-prop-2-ynyl-1H-indol-3-ylmethyl)-pyrrolidine-1-carbonyl]-propylcarbamoyl}-ethyl)-carbamicacid tert-butyl ester (41)

To a solution of 40 (0.150 g, 0.319 mmol) in THF (2 mL) was addedpropargyl bromide [0.06 mL, 0.410 mmol, (80% wt/toluene)] followed byNaH [0.015 g, 0.410 mmol, (60% dispersion in mineral oil)]. The reactionmixture was stirred overnight at room temperature. Water (2 mL) wasadded to the reaction mixture and the product was extracted with ethylacetate (3×30 mL). The ethyl acetate extracts were washed with water,brine and dried over anhydrous Na₂SO₄, filtered, and concentrated. Thecrude product was purified by HPLC. ¹H NMR (CDCl₃, 300 MHz): δ8.0 (s,1H), 7.9 (d, J=9.9 Hz, 1H), 7.38 (d, J=9.9 Hz, 1H), 7.3-7.1 (m, 3H), 6.8(m, 1H), 4.8 (s, 2H), 4.62 (m 1H), 4.5-4.4 (m 1H), 4.4-4.0 (m, 2H),3.7-3.5 (m, 2H), 3.4 (m, 1H), 2.5 (m, 1H), 2.4 (s, 1H), 2.2-1.8 (m, 4H),1.48 (s, 9H), 1.35 (d, J=9.9 Hz, 3H), 1.05 (d, J=5.5 Hz, 3H), 0.95 (d,J=5.5 Hz, 3H) ppm.

B.{1-[1-(2-{1-[6-(3-{1-[2-(2-tert-Butoxycarbonylamino-propionylamino)-3-methyl-butyryl]-pyrrolidin-2-ylmethyl}-indol-1-yl)-hexa-2,4-diynyl]-1H-indol-3-ylmethyl}-pyrrolidine-1-carbonyl)-2-methyl-propylcarbamoyl]-ethyl}-carbamicacid tert-butyl ester (42)

To a solution of 41 (0.040 g, 0.077 mmol) in acetonitrile (2 mL), wasadded copper (II) acetate (0.070 g, 0.385 mmol) and the reaction mixturewas immersed in a preheated oil bath (˜100° C.) and refluxed for 5 min.Water was then added to the reaction mixture (2 mL) and the product wasextracted with EtOAc (3×30 mL). The organic extracts were washed withaqueous NH₄OH (5 mL), water, brine and dried over anhydrous Na₂SO₄,filitered, and concentrated to afford the crude product. ¹H NMR (CDCl₃,300 MHz): δ8.0 (s, 2H), 7.9 (d, J=9.9 Hz, 2H), 7.38 (d, J=9.9 Hz, 2H),7.3-7.1 (m, 6H), 6.8 (m, 2H), 4.8 (s, 4H), 4.62 (m 2H), 4.5-4.4 (m 2H),4.4-4.0 (m, 4H), 3.7-3.5 (m, 4H), 3.4 (m, 2H), 2.5 (m, 2H), 2.2-1.8 (m,8H), 1.48 (s, 18H), 1.35 (d, J=9.9H, 6H), 1.05 (d, J=5.5 Hz, 6H), 0.95(d, J=5.5 Hz, 6H) ppm.

C.2-Amino-N-[1-(2-{1-[6-(3-{1-[2-(2-amino-propionylamino)-3-methyl-butyryl]-pyrrolidin-2-ylmethyl}-indol-1-yl)-hexa-2,4-diynyl]-1H-indol-3-ylmethyl}-pyrrolidine-1-carbonyl)-2-methyl-propyl]-propionamide(43)

To a solution of 42 (0.030 g, 0.029 mmol) in DCM (5 mL) was added TFA (1mL) and the reaction mixture was stirred at room temperature for 30 min.Aqueous NaHCO₃ (3 mL) was then added to the reaction mixture. Thereaction mixture was concentrated, diluted with water, and the productwas extracted with DCM (3×30 mL). The organic extracts were washed withwater, brine and dried over anhydrous Na₂SO₄. The solvent was removed onrotary evaporator and the product was purified by reverse phase HPLC. ¹HNMR (DMSO, 300 MHz) δ8.0 (s, 2H), 7.9 (d, J=9.9 Hz, 2H), 7.38 (d, J=9.9Hz, 2H), 7.3-7.1 (m, 6H), 6.8 (m, 2H), 4.8 (s, 4H), 4.62 (m 2H), 4.5-4.4(m 2H), 4.4-4.0 (m, 4H), 3.7-3.5 (m, 4H), 3.4 (m, 2H), 2.5 (m, 2H),2.2-1.8 (m, 8H), 1.35 (d, J=9.9 Hz, 6H), 1.05 (d, J=5.5 Hz, 6H), 0.95(d, J=5.5 Hz, 6H) ppm.

EXAMPLES

Example 8

Wherein R7a and R7b are independently H, alkyl, cycloalkyl, haloalkyl;or R8a and R7a and R8b and R7b can independently or together form a ringsuch as an aziridine or azetidine ring;

R8a and R8b are independently H, hydroxyl, alkyl, aryl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein eachalkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, andheteroarylalkyl is optionally-substituted with halogen, hydroxyl,mercapto, carboxyl, alkyl, alkoxy, amino, and nitro; or R8a and R7a andR8b and R7b can independently or together form a ring such as anaziridine or azetidine ring;

R5a and R5b are independently H, alkyl, cycloalkyl, cycloalkylalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl; or eachoptionally-substituted with hydroxyl, mercapto, halogen, amino,carboxyl, alkyl, haloalkyl, alkoxy, or alkylthio; or, optionally, R5aand R5b are connected by an alkylene, alkenylene, alkynylene of 2 to 12carbon atoms or optionally-substituted alkylene, allcenylene, alkynylenebridge of 2 to 12 carbon atoms where one or more carbon atoms can bereplaced with N, O, or S;

R12a, R12b, R13a, R13b, R14a, and R14b are independently H, Cl, Br, F,alkyl, cycloalkyl, hydroxy, alkoxy, amino, alkylamino, cyano, or CO₂H;

R3a and R3b are independently H, halogen, alkyl, aryl, arylalkyl, amino,arylamino, arylalkylamino, hydroxy, alkyloxy, aryloxy, arylalkylhydroxy,dialkylamino, amido, sulfonamido, or amidino; and

Wa and Wb are independently H, Cl, Br, F, alkyl, CN, or CO₂H.

Stereochem at Position Entry R8a R7a R5a (*) Wa Wb R3a R3b R5b R7b R8bR12 R13 R14 K_(D) Range 119 Me S-Me S-tBu S H H H H S-tBu S-Me Me H H HA 120 Me S-Me S-cHex S H H H H S-cHex S-Me Me H H H A 121 Me S-Me S—iPrS H H H H S—iPr S-Me Me H H H A

Preparation ofN-{1-Cyclohexyl-2-[2-(1-{2-[2-(3-{1-[2-cyclohexyl-2-(2-methylamino-propionylamino)-acetyl]-pyrrolidin-2-ylmethyl}-indol-1-yl)-ethoxy]-ethyl}-1H-indol-3-ylmethyl)-pyrrolidin-1-yl]-2-oxo-ethyl}-2-methylamino-propionamide(51)

Compound (46):

At 0° C., NaH (60%, 0.025 g, 0.62 mmol) was added to a solution ofindole 45 (0.17 g, 0.56 mmol) in anhydrous DMF (5 mL). After 1 h,bromoethyl ether (0.16 g, 0.68 mmol) and n-Bu₄NCl (0.021 g, 0.05 mmol)were added in rapid succession. The reaction mixture was allowed toslowly warm to ambient temperature and stirring was continued for 16 h.The reaction was quenched by the addition of saturated aqueous NH₄Cl andthe product was extracted with diethyl ether. The combined etherextracts were washed repeatedly with water to remove excess DMF thenbrine and dried over anhydrous Na₂SO₄, filtered, and concentrated. Thecrude product was combined with material formed under similar reactionconditions (0.11 g 45, 0.36 mmol) and purified by normal phase HPLC(10-100% EtOAc/hexane) to afford) 0.18 g of 46 as a colorless oil and0.18 g of the mono-alkylated indole. ¹H NMR (CDCl₃, 300 MHz) δ7.75-7.66(m, 2H), 7.25-7.07 (m, 6H), 6.85-6.80 (m, 2H), 4.13 (m, 8H), 3.62 (br s,4H), 3.40-3.15 (m, 4H), 2.63 (m, 2H), 2.04 (br s, 4H), 1.53 (s, 18H)ppm.

Compound (47):

A solution containing 46 (0.18 g, 0.27 mmol) in DCM (8 mL) was cooled to0° C. Trifluoroacetic acid (2 mL) was added and the reaction mixture wasmaintained at 0° C. for 1 h. The reaction was quenched by the carefuladdition of saturated aqueous NaHCO₃ and the product was extracted withEtOAc. The combined organic extracts were washed with aq. NaHCO₃, brine,dried over anhydrous Na₂SO₄, filtered, and concentrated to afford 0.075g of 47 as a pale yellow oil. The crude product was used directly in thenext reaction. ¹H NMR (CDCl₃, 300 MHz) δ8.49 (br s, 2H), 7.57 (app. d,J=8.2, Hz, 2H), 7.26-7.052 (m, 6H), 6.82 (s, 2H), 4.11 (m, 8H), 3.57 (m,4H), 3.29-2.96 (m, 6H), 1.95-1.58 (m, 8H) ppm.

2-(2-{1-[2-(2-{3-[1-(2-tert-Butoxycarbonylamino-2-cyclohexyl-acetyl)-pyrrolidin-2-ylmethy]-indol-1-yl}-ethoxy)-ethyl]-1H-indol-3-ylmethyl}-pyrrolidin-1-yl)-1-cyclohexyl-2-oxo-ethyl]carbamicacid tert-butyl ester (48)

To a solution containing N-Boc-cyclohexylglycine (0.09 g, 0.34 mmol) inanhydrous NMP (2 mL) was added HATU (0.15 g, 0.38 mmol) andN-methylmorpholine (0.042 g, 0.42 mmol). After 15 min, 47 (0.075 g, 0.16mmol) in anhydrous NMP (2 mL) was added and the reaction mixture wasstirred for 16 h. The reaction mixture was diluted with water and theproduct was extracted with diethyl ether. The combined ether extractswere washed repeatedly with water to remove excess NMP, brine, driedover anhydrous Na₂SO₄, filtered, and concentrated. The crude product waspurified by normal phase HPLC (50-100% EtOAc/hexane) to afford 0.085 mgof 48 as a colorless oil. ¹H NMR (CDCl₃, 300 MHz) δ7.87 (d, J=7.62 Hz,2H), 7.27-7.10 (m, 6H), 6.84 (s, 2H), 5.36 (d, J=9.37 Hz, 2H), 4.45 (m,2H), 4.30 (app. t, J=6.4 Hz, 2H), 4.18-4.12 (m, 6H), 3.70-3.58 (m, 6H),3.35 (dd, J=14.0, 2.9 Hz, 2H), 2.41 (dt, J=11.1, 2.3 Hz, 2H), 2.04-1.57(m, 20H), 1.44 (s, 18H), 1.37-1.07 (m, 10H) ppm.

2-Amino-1-(2-{1-[2-(2-{3-[1-(2-amino-2-cyclohexyl-acetyl)-pyrrolidin-2-ylmethyl]-indol-1-yl}-ethoxy)-ethyl]-1H-indol-3-ylmethyl}-pyrrolidin-1-yl)-2-cyclohexyl-ethanone(49)

A solution containing 48 (0.085 g, 0.08 mmol) in DCM (8 mL) was cooledto 0° C. Trifluoroacetic acid (2 mL) was added and the reaction mixturewas maintained at 0° C. for 30 min. An additional portion of TFA (1 mL)was added and the reaction mixture was stirred for 1 h at 0° C. Thereaction was quenched by the careful addition of saturated aqueousNaHCO₃ and the product was extracted with EtOAc. The combined organicextracts were washed with aq. NaHCO₃, brine, dried over anhydrousNa₂SO₄, filtered, and concentrated to afford 0.068 g of 49 as a paleyellow oil. The crude product was used directly in the next reaction. ¹HNMR (CDCl₃, 300 MHz) δ7.90 (d, J=7.6 Hz, 2H), 7.31-7.12 (m, 6H), 6.84(app t, J=14 Hz, 2H), 4.47 (m, minor rotomer), 4.15 (m, 4H), 3.66-3.36(m, 8H), 2.80 (m, 2H), 2.42 (m, 2H), 2.04-0.83 (m, 30H) ppm.

Amide (50):

To a solution containing N-Boc-N-methylalanine (0.041 g, 0.19 mmol) inanhydrous NMP (2 mL) was added HATU (0.083 g, 0.21 mmol) andN-methylmorpholine (0.024 g, 0.23 mmol). After 15 min, 49 (0.068 g, 0.09mmol) in anhydrous NMP (2 mL) was added and the reaction mixture wasstirred for 16 h. The reaction mixture was diluted with water and theproduct was extracted with diethyl ether. The combined ether extractswere washed repeatedly with water to remove excess NMP, brine, driedover anhydrous Na₂SO₄, filtered, and concentrated. The crude product wascombined with material formed under similar reaction conditions (0.05 g49, 0.06 mmol) and purified by normal phase HPLC (10-100% EtOAc/hexane)to afford 32 mg of 50 as a colorless oil. ¹H NMR (CDCl₃, 300 MHz) δ7.86(d, J=7.6 Hz, 2H), 7.27-7.08 (m, 6H), 6.84 (s, 2H), 4.70 (m, 2H), 4.58(app. t, J=7.6 Hz, 2H), 4.19 (m, 2H), 4.15-4.04 (m, 6H), 3.76-3.60 (m,6H), 3.37 (m, 2H), 2.83 (s, 6H), 2.42 (m, 2H), 1.98-1.55 (m, 20H), 1.51(s, 18H), 1.33 (d, J=7.0 Hz, 6H), 1.30-0.98 (m, 10H) ppm.

N-{1-Cyclohexyl-2-[2-(1-{2-[2-(3-{1-[2-cyclohexyl-2-(2-methylamino-propionylamino)-acetyl]-pyrrolidin-2-ylmethyl}-indol-1-yl)-ethoxy]-ethyl}-1H-indol-3-ylmethyl)-pyrrolidin-1-yl]-2-oxo-ethyl}-2-methylamino-propionamide(51)

A solution containing 50 (0.0.062 g, 0.055 mmol) in DCM (8 mL) wascooled to 0° C. Trifluoroacetic acid (2 mL) was added and the reactionmixture was maintained at 0° C. for 1 h. The reaction was quenched bythe careful addition of saturated aqueous NaHCO₃ and the product wasextracted with EtOAc. The combined organic extracts were washed with aq.NaHCO₃, brine, dried over anhydrous Na₂SO₄, filtered, and concentrated.The crude product was purified by reverse phase HPLC (10-100% ACN/waterw/0.1% HOAc) to afford 0.047 g of 51.2HOAc as a white solid followinglyophilization. ¹H NMR (DMSO, 300 MHz) δ7.91 (d, J=8.7 Hz, 2H), 7.71 (d,J=7.8 Hz, 2H), 7.32 (d, J=7.5 Hz, 2H), 7.05 (m, 2H), 6.99-6.92 (m, 4H),4.39 (app. t, J=6.6 Hz, 2H), 4.15 (m, 6H), 3.57 (m, 4H), 3.52-3.23 (m,4H), 3.06-2.91 (m, 4H), 2.46 (s, 6H), 2.31 (m, 2H), 2.15 (s, 12H),1.91-1.65 (m, 12H), 1.55-1.49 (m, 8H), 1.07 (d, J=7.0 Hz, 6H), 1.16-0.93(m, 10H) ppm.

EXAMPLES

Example 9

Wherein R7a and R7b are independently H, alkyl, cycloalkyl, haloalkyl;or R8a and R7a and R8b and R7b can independently or together form a ringsuch as an aziridine or azetidine ring;

R8a and R8b are independently H, hydroxyl, alkyl, aryl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein eachalkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, andheteroarylalkyl is optionally-substituted with halogen, hydroxyl,mercapto, carboxyl, alkyl, alkoxy, amino, and nitro; or R8a and R7a andR8b and R7b can independently or together form a ring such as anaziridine or azetidine ring;

R5a and R5b are independently H, alkyl, cycloalkyl, cycloalkylalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl; or eachoptionally-substituted with hydroxyl, mercapto, halogen, amino,carboxyl, alkyl, haloalkyl, alkoxy, or alkylthio; or, optionally, R5aand R5b are connected by an alkylene, alkenylene, alkynylene of 2 to 12carbon atoms or optionally-substituted alkylene, alkenylene, alkynylenebridge of 2 to 12 carbon atoms where one or more carbon atoms can bereplaced with N, O, or S;

R12a, R12b, R13a, R13b, R14a, and R14b are independently H, Cl, Br, F,alkyl, cycloalkyl, hydroxy, alkoxy, amino, alkylamino, cyano, or CO₂H;

R3a and R3b are independently H, halogen, alkyl, aryl, arylalkyl, amino,arylamino, arylalkylamino, hydroxy, alkyloxy, aryloxy, arylalkylhydroxy,dialkylamino, amido, sulfonamido, or amidino; and

Wa and Wb are independently H, Cl, Br, F, alkyl, CN, CO₂H.

Stereochem at Position Entry R8a R7a R5a (*) Wa Wb R3a R3b R5b R7b R8bR12 R13 R14 K_(D) Range, 122 Me S-Me S-cHex S H H H H S-cHex S-Me Me H HH A 123 Me S-Me S-cHex S H H H H S-cHex S-Me Me H F H A 124 Me S-MeS-(2R-EtOH) S H H H H S-(2R-EtOH) S-Me Me H F H A 125 Me S-EtS-(2R-EtOH) S H H H H S-(2R-EtOH) S-Et Me F H H B 126 Me S-Me S-cHex S HH H H S-cHex S-Me Me F H H A 127 Me S-Me S-cHex S Me Me H H S-cHex S-MeMe F H H A 128 Me S-Et S-cHex S Me Me H H S-cHex S-Et Me F H H A 129 HS-Me S-cHex S H H H H S-cHex S-Me H Cl H H A 130 Me S-Me S-cHex S H H HH S-cHex S-Me Me Cl H H A 131 H S-Me S—iPr S H H H H S—iPr S-Me H Cl H HA 132 Me S-Me S—iPr S H H H H S—iPr S-Me Me Cl H H A 133 Me S-Me S—iPr SH H H H S—iPr S-Me Me F H H A 134 H H S-cHex S H H H H S-cHex H H H F HC 135 Me S-Me H S H H H H H S-Me Me H F H C 136 Me S-Et S-(2R-EtOH) S HH H H S-(2R-EtOH) S-Et Me H F H C 137 Me S-Me S-cHex S H H H H S-cHexS-Me Me Me H H A 138 H H S-cHex S H H H H S-cHex H H H H H B 139 H S-MeS-cHex S H H H H S-cHex S-Me H H H H A 140 Me S-Me S-tBu S H H H H S-tBuS-Me Me H F H A 141 Me S-Me S-tBu S H H H H S-tBu S-Me Me H H H A 142 HS-Me S-tBu S H H H H S-tBu S-Me H H H H A 143 Me S-Me S-cHex S H H H HS-cHex S-Me H H H H A 144 Me S-Me S-cHex S H H H H S-cHex H H H H H A145 Me S-Me S-(2R-EtOH) S H H H H S-(2R-EtOH) S-Me Me H H H B 146 MeS-Me S-(CH₂)₄NH₂ S H H H H S-(CH₂)₄NH₂ S-Me Me Me H H A

Example 10

Wherein R7a and R7b are independently H, alkyl, cycloalkyl, haloalkyl;or R8a and R7a and R8b and R7b can independently or together form a ringsuch as an aziridine or azetidine ring;

R8a and R8b are independently H, hydroxyl, alkyl, aryl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein eachalkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, andheteroarylalkyl is optionally-substituted with halogen, hydroxyl,mercapto, carboxyl, alkyl, alkoxy, amino, and nitro; or R8a and R7a andR8b and R7b can independently or together form a ring such as anaziridine or azetidine ring;

R5a and R5b are independently H, alkyl, cycloalkyl, cycloalkylalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl; or eachoptionally-substituted with hydroxyl, mercapto, halogen, amino,carboxyl, alkyl, haloalkyl, alkoxy, or alkylthio; or, optionally, R5aand R5b are connected by an alkylene, alkenylene, alkynylene of 2 to 12carbon atoms or optionally-substituted alkylene, alkenylene, alkynylenebridge of 2 to 12 carbon atoms where one or more carbon atoms can bereplaced with N, O, or S;

R12a, R12b, R13a, R13b, R14a, and R14b are independently H, Cl, Br, F,alkyl, cycloalkyl, hydroxy, alkoxy, amino, alkylamino, cyano, or CO₂H;

R3a and R3b are independently H, halogen, alkyl, aryl, arylalkyl, amino,arylamino, arylalkylamino, hydroxy, alkyloxy, aryloxy, arylalkylhydroxy,dialkylamino, amido, sulfonamido, or amidino;

Wa and Wb are independently H, Cl, Br, F, alkyl, CN, CO₂H; and

R11a and R11b together form an alkylene, alkenylene, alkynlyene, oralkyloxyalkylene chain of 2 to 12 carbon atoms or optionally-substitutedalkylene, alkenylene, alkynlyene, or alkyloxyalkylene chain of 2 to 12carbon atoms where one or more carbon atoms can be replaced with N, O,or S.

Stereochem at Position Entry R8a R7a R5a Wa Wb (*) R11a-R11b 147 H S-MeS—iPr H H S CH₂CH₂CH₂CH₂CH₂CH₂ 148 Me S-Me S-cHex H H SCH₂CH₂CH₂CH₂CH₂CH₂ 149 Me S-Me S-tBu H H S CH₂CH₂CH₂CH₂CH₂CH₂ 150 H S-MeiPr H H S (R,R)-CH₂CH(OH)CH(OH)CH₂ 151 Me S-Me 2R-EtOH H H S(R,R)-CH₂CH(OH)CH(OH)CH₂ 152 Me S-Me 2R-EtOH H H S(S,S)-CH₂CH(OH)CH(OH)CH₂ 153 Me S-Me cHex H H S CH₂CH₂CH₂ 154 Me S-MecHex H H S CH₂CH₂OCH₂CH₂OCH₂CH₂ 155 Me S-Et cHex H H SCH₂CH₂OCH₂CH₂OCH₂CH₂ 156 Me S-Me 2R-EtOH H H S CH₂CH₂OCH₂CH₂OCH₂CH₂ 157Me S-Et 2R-EtOH H H S CH₂CH₂OCH₂CH₂OCH₂CH₂ 158 Me S-Me cHex H H SC(O)CH₂CH₂CH₂C(O) 159 Me S-Me cHex H H S C(O)C₆H₄C(O) 160 Me S-Me2R-EtOH H H S CH₂CH₂ 161 Me S-Me 2R-EtOH H H S CH₂CH₂CH₂CH₂ Entry R3aR3b R5b R7b R8b R12 R13 R14 K_(D) Range, 147 H H S—iPr S-Me H H H H A148 H H S-cHex S-Me Me H H H A 149 H H S-tBu S-Me Me H H H A 150 H H iPrS-Me H H F H A 151 H H 2R-EtOH S-Me Me H F H A 152 H H 2R-EtOH S-Me Me HF H A 153 H H cHex S-Me Me H H H B 154 H H cHex S-Me Me H H H A 155 H HcHex S-Et Me H H H B 156 H H 2R-EtOH S-Me Me H H H A 157 H H 2R-EtOHS-Et Me H H H B 158 H H cHex S-Me Me H H H A 159 H H cHex S-Me Me H H HA 160 H H 2R-EtOH S-Me Me H H H A 161 H H 2R-EtOH S-Me Me H H H B

Further Examples

Example 11

Wherein R7a and R7b are independently H, alkyl, cycloalkyl, haloalkyl;or R8a and R7a and R8b and R7b can independently or together form a ringsuch as an aziridine or azetidine ring;

R8a and R8b are independently H, hydroxyl, alkyl, aryl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein eachalkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, andheteroarylalkyl is optionally-substituted with halogen, hydroxyl,mercapto, carboxyl, alkyl, alkoxy, amino, and nitro; or R8a and R7a andR8b and R7b can independently or together form a ring such as anaziridine or azetidine ring;

R5a and R5b are independently H, alkyl, cycloalkyl, cycloalkylalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl; or eachoptionally-substituted with hydroxyl, mercapto, halogen, amino,carboxyl, alkyl, haloalkyl, alkoxy, or alkylthio; or, in some instances,the R5a and R5b residues are connected by an alkylene, alkenylene,alkynylene of 2 to 12 carbon atoms or optionally-substituted alkylene,alkenylene, alkynylene bridge of 2 to 12 carbon atoms where one or morecarbon atoms can be replaced with N, O, or S;

R12a, R12b, R13a, R13b, R14a, and R14b are independently H, Cl, Br, F,alkyl, cycloalkyl, hydroxy, alkoxy, amino, alkylamino, cyano, or CO₂H;

R3a and R3b are independently H, halogen, alkyl, aryl, arylalkyl, amino,arylamino, arylalkylamino, hydroxy, alkyloxy, aryloxy, arylalkylhydroxy,dialkylamino, amido, sulfonamido, or amidino;

X is O, N, S, or

Wa is H, Cl, Br, F, alkyl, CN, CO₂H;

R11b is absent or H, alkyl, optionally-substituted alkyl, hydroxyalkyl,alkoxyalkyl.

Wb and R11a together are a bond, alkylene, alkenylene, alkynylene, aryl,arylalkylene, arylalkylalkylene, heteroaryl, heteroarylalkylene, or anoptionally-substituted alkylene, alkenylene, alkynylene chain of 2 to 12carbon atoms where one or more carbon atoms can be replaced with N, O,or S.

Stereochem at Position K_(D) Entry R8a R7a R5a Wa R11b (*) X Wb-R11a R3aR3b R5b R7b R8b R12 R13 R14 Pανγε 162 Me S-Me S-(2R- H - S O CH₂CH₂CH₂ HH S-(2R-EtOH S-Me Me H H H D EtOH) 163 Me S-Me S-cHex H - S O CH₂CH₂CH₂H H S-cHex S-Me Me H H H A

In mammalian cells activation of the caspases is achieved through atleast two independent mechanisms which are initiated by distinctcaspases, but result in the activation of common executioner (effector)caspases. In addition to the cytochrome c activated mechanism (sometimesreferred to as the ‘intrinsic death pathway’), the ‘extrinsic deathpathway’ is a mechanism by which the caspase cascade is activated viaactivation of a death receptor located on the cell membrane. Examples ofdeath receptors include DR4, DR5 and TNF-R1 (as well as other members ofthe TNF group of cytokine receptors). The corresponding ligands areTRAIL and TNF-α, respectfully. Binding of pro-caspase-8 to the deathreceptor induces auto-activation wherein the inhibitory pro-domain ofpro-caspase-8 is cleaved and removed. Caspase-8 is released from thereceptor and can then activate effector caspases (caspase-3, -6, -7),and as in the caspase-9 initiated pathway, the result is the proteolyticcleavage of cellular targets by the effector caspases and the inductionof apoptosis.

The present invention is directed generally to Smac peptidomimetics,methods of making Smac peptidomimetics and uses thereof includingmethods of making the peptidomimetics described above. In one embodimentof the current invention, Smac peptidomimetics (herein referred to asSmac mimetic) act as chemopotentiating agents. The term“chemopotentiating agent” refers to an agent that acts to increase thesensitivity of an organism, tissue, or cell to a chemical compound, ortreatment namely “chemotherapeutic agents” or “chemo drugs” or radiationtreatment. One embodiment of the invention is the therapeuticcomposition of a Smac mimetic. A further embodiment of the invention isthe therapeutic composition of a Smac mimetic, which can act as achemopotentiating agent and a biological or chemotherapeutic agent orradiation. Another embodiment of the invention is a method of inhibitingtumor growth in vivo by administering a Smac peptidomimetic. Anotherembodiment of the invention is a method of inhibiting tumor growth invivo by administering a Smac mimetic and a biologic or chemotherapeuticagent or chemoradiation. Another embodiment of the invention is a methodof treating a patient with a cancer by administering Smac mimetics ofthe present invention alone or in combination with a biological orchemotherapeutic agent or chemoradiation.

In a preferred embodiment of the present invention, suitable biologicaland chemotherapeutic agents that can be administered concurrently withSmac mimetics include alkylating agents, plant alkaloids, anti-tumorantibiotics, antimetabolites, topoisomerase inhibitors, hormonal agents,NSAIDs, growth factors, cytokines, mitotic inhibitors and combinationsof these.

In another embodiment of the present invention, the cells are in situ orin an individual, and the contacting step is affected by administering apharmaceutical composition comprising a therapeutically effective amountof the Smac mimetic wherein the individual may be subject to concurrentor antecedent radiation or chemotherapy for treatment of aneoproliferative pathology. Pathogenic cells are of a tumor such as, butnot limited to, bladder cancer, breast cancer, prostate cancer, lungcancer, pancreatic cancer, gastric cancer, colon cancer, ovarian cancer,renal cancer, hepatoma, melanoma, lymphoma, sarcoma, and combinationsthereof. However, the cells may also be immortalized tumor cells used intumor cell culture.

Smac mimetics may also be used to treat autoimmune diseases. In additionto apoptosis defects found in tumors, defects in the ability toeliminate self-reactive cells of the immune system due to apoptosisresistance are considered to play a key role in the pathogenesis ofautoimmune diseases. Autoimmune diseases are characterized in that thecells of the immune system produce antibodies against its own organs andmolecules or directly attack tissues resulting in the destruction of thelatter. A failure of those self-reactive cells to undergo apoptosisleads to the manifestation of the disease. Defects in apoptosisregulation have been identified in autoimmune diseases such as systemiclupus erythematosus or rheumatoid arthritis.

In one embodiment the pathogenic further include abnormallyproliferating cells such as those of any autoimmune disease or diseaseswhich are resistant to apoptosis due to the overexpression of IAPs ormembers of the Bcl-2 family of proteins. Examples of such autoimmunediseases include but are not limited to collagen diseases such asrheumatoid arthritis, systemic lupus erythematosus, Sharp's syndrome,CREST syndrome (calcinosis, Raynaud's syndrome, esophageal dysmotility,telangiectasia), dermatomyositis, vasculitis (Morbus Wegener's) andSjögren's syndrome, renal diseases such as Goodpasture's syndrome,rapidly-progressing glomerulonephritis and membrano-proliferativeglomerulonephritis type II, endocrine diseases such as type-I diabetes,autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),autoimmune parathyroidism, pernicious anemia, gonad insufficiency,idiopathic Morbus Addison's, hyperthyreosis, Hashimoto's thyroiditis andprimary myxedema, skin diseases such as pemphigus vulgaris, bullouspemphigoid, herpes gestationis, epidermolysis bullosa and erythemamultiforme major, liver diseases such as primary biliary cirrhosis,autoimmune cholangitis, autoimmune hepatitis type-1, autoimmunehepatitis type-2, primary sclerosing cholangitis, neuronal diseases suchas multiple sclerosis, myasthenia gravis, myasthenic Lambert-Eatonsyndrome, acquired neuromyotony, Guillain-Barré syndrome (Müller-Fischersyndrome), stiff-man syndrome, cerebellar degeneration, ataxia,opsoklonus, sensoric neuropathy and achalasia, blood diseases such asautoimmune hemolytic anemia, idiopathic thrombocytopenic purpura (MorbusWerlhof), infectious diseases with associated autoimmune reactions suchas AIDS, Malaria and Chagas disease.

Pharmaceutical compositions encompassed by the present invention includea therapeutically effective amount of a Smac mimetic in dosage form anda pharmaceutically acceptable carrier, wherein the Smac mimetic inhibitsthe activity of an IAP, thus, promoting apoptosis. Another embodiment ofthe present invention are compositions containing a therapeuticallyeffective amount of a Smac mimetic in dosage form and a pharmaceuticallyacceptable carrier, in combination with a biological or chemotherapeuticagent and/or radiotherapy, wherein the Smac mimetic inhibits theactivity of an IAP, thus, promoting apoptosis and enhancing theeffectiveness of the chemotherapeutic and/or radiotherapy.

Methods of making pharmaceutical compositions containing Smac mimeticsare also encompassed in the present invention and include but are notlimited to combining a therapeutically effective amount of the Smacmimetic with a pharmaceutically acceptible exipient.

In an embodiment of the invention a therapeutic composition forpromoting apoptosis a therapeutically effective amount of a Smacpeptidomimetic that binds to at least one IAP. In one embodiment the IAPcan be XIAP. In another embodiment the IAP can be ML-IAP. In anotherembodiment the IAP can be cIAP-1 or cIAP-2. In a further embodiment theIAP can be multiple IAP types.

Embodiments of the invention also include a method of treating a patientwith a condition in need thereof wherein administration of atherapeutically effective amount of a Smac peptidomimetic is deliveredto the patient, and the Smac peptidomimetic binds to at least one IAP.In one embodiment the IAP can be XIAP. In another embodiment the IAP canbe ML-IAP. In another embodiment the IAP can cIAP-1 or cIAP-2. In afurther embodiment the IAP can be multiple IAP types. The method mayfurther include the concurrent administration chemotherapeutic agent.The chemotherapeutic agent can be, but is not limited to, alkylatingagents, antimetabolites, anti-tumor antibiotics, taxanes, hormonalagents, monoclonal antibodies, glucocorticoids, mitotic inhibitors,topoisomerase I inhibitors, topoisomerase II inhibitors,immunomodulating agents, cellular growth factors, cytokines, andnonsteroidal anti-inflammatory compounds.

Smac mimetics are preferably administered in effective amounts. Aneffective amount is that amount of a preparation that alone, or togetherwith further doses, produces the desired response. This may involve onlyslowing the progression of the disease temporarily, although preferably,it involves halting the progression of the disease permanently ordelaying the onset of or preventing the disease or condition fromoccurring. This can be monitored by routine methods. Generally, doses ofactive compounds would be from about 0.01 mg/kg per day to 1000 mg/kgper day. It is expected that doses ranging from 50-500 mg/kg will besuitable, preferably intravenously, intramuscularly, or intradermally,and in one or several administrations per day. The administration of theSmac peptidomimetic can occur simultaneous with, subsequent to, or priorto chemotherapy or radiation so long as the chemotherapeutic agent orradiation sensitizes the system to the Smac peptidomimetic.

In general, routine experimentation in clinical trials will determinespecific ranges for optimal therapeutic effect for each therapeuticagent and each administrative protocol, and administration to specificpatients will be adjusted to within effective and safe ranges dependingon the patient condition and responsiveness to initial administrations.However, the ultimate administration protocol will be regulatedaccording to the judgment of the attending clinician considering suchfactors as age, condition and size of the patient, the Smacpeptidomimetic potencies, the duration of the treatment and the severityof the disease being treated. For example, a dosage regimen of the Smacpeptidomimetic can be oral administration of from 1 mg to 2000 mg/day,preferably 1 to 1000 mg/day, more preferably 50 to 600 mg/day, in two tofour (preferably two) divided doses, to reduce tumor growth.Intermittent therapy (e.g., one week out of three weeks or three out offour weeks) may also be used.

In the event that a response in a subject is insufficient at the initialdoses applied, higher doses (or effectively higher doses by a different,more localized delivery route) may be employed to the extent that thepatient tolerance permits. Multiple doses per day are contemplated toachieve appropriate systemic levels of compounds. Generally, a maximumdose is used, that is, the highest safe dose according to sound medicaljudgment. Those of ordinary skill in the art will understand, however,that a patient may insist upon a lower dose or tolerable dose formedical reasons, psychological reasons or for virtually any otherreason.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular chemotherapeuticdrug selected, the severity of the condition being treated and thedosage required for therapeutic efficacy. The methods of the invention,generally speaking, may be practiced using any mode of administrationthat is medically acceptable, meaning any mode that produces effectivelevels of the active compounds without causing clinically unacceptableadverse effects. Such modes of administration include, but are notlimited to, oral, rectal, topical, nasal, intradermal, inhalation,intra-peritoneal, intravesical or parenteral routes. The term“parenteral” includes subcutaneous, intravenous, intramuscular, orinfusion. Intravenous or intramuscular routes are particularly suitablefor purposes of the present invention.

In one aspect of the invention, a Smac peptidomimetic as describedherein, with or without additional biological or chemotherapeutic agentsor radiotherapy, does not adversely affect normal tissues, whilesensitizing tumor cells to the additional chemotherapeutic/radiationprotocols. While not wishing to be bound by theory, it would appear thatbecause of this tumor specific induced apoptosis, marked and adverseside effects such as inappropriate vasodilation or shock are minimized.Preferably, the composition or method is designed to allow sensitizationof the cell or tumor to the chemotherapeutic or radiation therapy byadministering at least a portion of the Smac peptidomimetic prior tochemotherapeutic or radiation therapy. The radiation therapy, and/orinclusion of chemotherapeutic agents, may be included as part of thetherapeutic regimen to further potentiate the tumor cell killing by theSmac peptidomimetic.

In alternative embodiments of the current invention, Smac mimetics areadministered in comabination with a second form of therapy including butnot limited to second therapy selected from radiation therapy,immunotherapy, photodynamic therapy, chemotherapy and combinationsthereof.

Anti-cancer chemotherapeutic agents administered in combination withSmac mimetics may be any therapeutic agent that specifically targetstumorigenic tissue or cells and include but is not limited to alkylatingagents, plant alkaloids, antitumor antibiotics, antimetabolites, andtopoisomerase inhibitors including altretamine, busulfan, carboplatin,carmustine, chlorambucil, cisplatin, cyclophosphomide, dacarbazine,hexamethylmelamine, ifosfamide, lomustine, melphalan, mechlorethamine,oxaliplatin, procarbazine, streptozocin, temozolomide, thiotepa,uramustine, docetaxel, etoposide, irinotecan, paclitaxel, tenisopide,vincristine, vinblastine, vindesine, vinorelbine, bleomycin,dactinomycin, daunorubicin, epirubicin, hydroxyurea, idarubicin,mitomycin, mitoxantrone, plicamycin, azathioprine, capecitabine,cladribine, cytarabine, fludarabine, fluorouracil, floxuridine,gemcitabine, mercaptopurine, methotrexate, nelarabine, pemeterxed,pentostatin, thioguanine, camptothecan, topotecan, BNP 1350, SN 38,9-amino-camptothecan, lurtotecan, gimatecan, diflomotecan, doxorubicin,epirubicin, idarubicin, nemorubicin, mitoxantrone, loxoxantrone,etoposide, and combinations thereof.

Smac mimetics as described herein may also be administered concurrentlywith immunotherapeutic agents. Immunotherapy includes the administrationof an immunologically active agent selected from bacillusCalmette-Guerin (BCG), interferon, and other agents that specificallyactivate the immune system of affected patients and combinationsthereof.

Pharmaceutical Compositions.

In one embodiment of the invention, an additional biological,chemotherapeutic or anti-neoplastic agent (infra) and/or radiation maybe added prior to, along with, or following the administration of a Smacmimetic. The term “pharmaceutically-acceptable carrier” as used hereinmeans one or more compatible solid or liquid fillers, diluents orencapsulating substances which are suitable for administration into ahuman. The term “carrier” denotes an organic or inorganic ingredient,natural or synthetic, with which the active ingredient is combined tofacilitate the application. The components of the pharmaceuticalcompositions are capable of being co-mingled with the molecules of thepresent invention, and with each other, in a manner such that there isno interaction which would substantially impair the desiredpharmaceutical efficacy.

The delivery systems of the invention are designed to includetime-released, delayed release or sustained release delivery systemssuch that the delivering of the Smac peptidomimetic occurs prior to, andwith sufficient time, to cause sensitization of the site to be treated.A Smac peptidomimetic may be used in conjunction with radiation and/oradditional anti-cancer chemical agents (infra). Such systems can avoidrepeated administrations of the Smac peptidomimetc compound, increasingconvenience to the subject and the physician, and may be particularlysuitable for certain compositions of the present invention.

Many types of release delivery systems are available and known to thoseof ordinary skill in the art and may be used in the context of thepresent invention including but not limited to polymer base systems suchas poly(lactide-glycolide), copolyoxalates, polycaprolactones,polyesteramides, polyorthoesters, polyhydroxybutyric acid, andpolyanhydrides. Microcapsules of the foregoing polymers containing drugsare described in, for example, U.S. Pat. No. 5,075,109. Delivery systemsalso include non-polymer systems that are: lipids including sterols suchas cholesterol, cholesterol esters and fatty acids or neutral fats suchas mono-di- and tri-glycerides; hydrogel release systems; sylasticsystems; peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to: (a) erosionalsystems in which the active compound is contained in a form within amatrix such as those described in U.S. Pat. Nos. 4,452,775, 4,667,014,4,748,034 and 5,239,660 and (b) diffusional systems in which an activecomponent permeates at a controlled rate from a polymer such asdescribed in U.S. Pat. Nos. 3,832,253, and 3,854,480. In addition,pump-based hardware delivery systems can be used, some of which areadapted for implantation.

Use of a long-term sustained release implant may be desirable. Long-termrelease, are used herein, means that the implant is constructed andarranged to deliver therapeutic levels of the active ingredient for atleast 30 days, and preferably 60 days. Long-term sustained releaseimplants are well-known to those of ordinary skill in the art andinclude some of the release systems described above.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy. All methods include the step of bringing the activeagent into association with a carrier that constitutes one or moreaccessory ingredients or exipients. In general, the compositions areprepared by uniformly and intimately bringing the active compound intoassociation with a liquid carrier, a finely divided solid carrier, orboth, and then, if necessary, shaping the product.

In one embodiment of the invention, the dimeric peptidomimeticsdescribed above are combined with a pharmaceutically acceptableexipient.

Compositions suitable for parenteral administration conveniently includea sterile aqueous preparation of a chemopotentiating agent (e.g. Smacpeptidomimetic), which is preferably isotonic with the blood of therecipient. This aqueous preparation may be formulated according to knownmethods using suitable dispersing or wetting agents and suspendingagents. The sterile injectable preparation also may be a sterileinjectable solution or suspension in a non-toxic parenterally-acceptablediluent or solvent, for example, as a solution in 1,3-butane diol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose, any bland fixed oil may be employedincluding synthetic mono- or di-glycerides. In addition, fatty acidssuch as oleic acid may be used in the preparation of injectables.Carrier formulation suitable for oral, subcutaneous, intravenous,intramuscular, etc. administrations can be found in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa. which isincorporated herein in its entirety by reference thereto.

Additional Chemotherapeutic Agents.

Chemotherapeutic agents suitable for use in combination with the presentinvention, include but are not limited to the chemotherapeutic agentsdescribed in “Modern Pharmacology with Clinical Applications”, SixthEdition, Craig & Stitzel, Chpt. 56, pg 639-656 (2004), hereinincorporated by reference in its entirety. This reference describeschemotherapeutic drugs to include alkylating agents, antimetabolites,anti-tumor antibiotics, plant-derived products such as taxanes, enzymes,hormonal agents such as glucocorticoids, miscellaneous agents such ascisplatin, monoclonal antibodies, immunomodulating agents such asinterferons, and cellular growth factors. Other suitable classificationsfor chemotherapeutic agents include mitotic inhibitors and nonsteroidalanti-estrogenic analogs. Other suitable chemotherapeutic agents includetoposiomerase I and II inhibitors, kinase inhibitors and any agentcapable of activating the extrinsic or intrinsic apoptotic pathway orrelease of Smac from the mitochondria.

Specific examples of suitable biological and chemotherapeutic agentsinclude, but are not limited to, cisplatin, carmustine (BCNU),5-fluorouracil (5-FU), cytarabine (Ara-C), gemcitabine, methotrexate,daunorubicin, doxorubicin, dexamethasone, topotecan, etoposide,paclitaxel, vincristine, tamoxifen, TNF-alpha, TRAIL, interferon (inboth its alpha and beta forms), thalidomide, and melphalan. Otherspecific examples of suitable chemotherapeutic agents include nitrogenmustards such as cyclophosphamide, alkyl sulfonates, nitrosoureas,ethylenimines, triazenes, folate antagonists, purine analogs, pyrimidineanalogs, anthracyclines, bleomycins, mitomycins, dactinomycins,plicamycin, vinca alkaloids, epipodophyllotoxins, taxanes,glucocorticoids, L-asparaginase, estrogens, androgens, progestins,luteinizing hormones, octreotide actetate, hydroxyurea, procarbazine,mitotane, hexamethylmelamine, carboplatin, mitoxantrone, monoclonalantibodies, levamisole, interferons, interleukins, filgrastim andsargramostim. Chemotherapeutic compositions also include other members,i.e., other than TRAIL, of the TNF superfamily of compounds or agentssuch as BCG which induce synthesis of chemokines following intravesicaltreatment. NSAIDS may also be used in combination with the Smac mimeticsof the present invention and may include selective and non-selectiveCOX-2 inhibitors, celecoxib and rofecoib.

Radiotherapy Protocols.

Additionally, in several method embodiments of the present invention theSmac peptidomimetic therapy may be used in connection withchemo-radiation or other cancer treatment protocols used to inhibittumor cell growth. For example, but not limited to, radiation therapy(or radiotherapy) is the medical use of ionizing radiation as part ofcancer treatment to control malignant cells is suitable for use inembodiments of the present invention. Although radiotherapy is oftenused as part of curative therapy, it is occasionally used as apalliative treatment, where cure is not possible and the aim is forsymptomatic relief. Radiotherapy is commonly used for the treatment oftumors. It may be used as the primary therapy. It is also common tocombine radiotherapy with surgery and/or chemotherapy. The most commontumors treated with radiotherapy are breast cancer, prostate cancer,rectal cancer, head & neck cancers, gynecological tumors, bladder cancerand lymphoma. Radiation therapy is commonly applied just to thelocalized area involved with the tumor. Often the radiation fields alsoinclude the draining lymph nodes. It is possible but uncommon to giveradiotherapy to the whole body, or entire skin surface. Radiationtherapy is usually given daily for up to 35-38 fractions (a daily doseis a fraction). These small frequent doses allow healthy cells time togrow back, repairing damage inflicted by the radiation. Three maindivisions of radiotherapy are external beam radiotherapy or teletherapy,brachytherapy or sealed source radiotherapy and unsealed sourceradiotherapy, which are all suitable examples of treatment protocol inthe present invention. The differences relate to the position of theradiation source; external is outside the body, while sealed andunsealed source radiotherapy has radioactive material deliveredinternally. Brachytherapy sealed sources are usually extracted later,while unsealed sources are injected into the body. Administration of theSmac peptidomimetic may occur prior to, concurrently with the treatmentprotocol.

The relative binding affinity of a Smac tetrapeptide (AVPI) and a potentSmac mimetic (Entry 17) to XIAP BIR-3 is shown in FIG. 1 This figurereveals the marked increase in binding affinity, a 30,000 fold increase,of the Smac mimetic Entry 17 relative to the Smac tetrapeptide.

The half life of 3 Smac mimetics, Entry 1, Entry 122 and Entry 123, wasexamined in a rat. The IV dose for each Smac mimetic was 1 mg/kg. FIG. 2shows that the terminal elimination half life is up to approximately 6hours for the Smac mimetics, with Entry 1 having the longest half-life.

Biological and chemotherapeutics/anti-neoplastic agents and radiationinduce apoptosis by activating the extrinsic or intrinsic apoptoticpathways, and since Smac mimetics relieve inhibitors of apoptoticproteins (IAP's) and, thus, remove the block in apoptosis, thecombination of chemotherapeutics/anti-neoplastic agents and radiationwith Smac mimetics should work synergistically to facilitate apoptosis.To show the synergistic effects of Smac mimetics with commonchemotherapeutic agents, a panel of diverse tumor cell lines wasselected and representative compounds from various mechanistic classesof chemotherapeutics, as well as gamma radiation were tested.

A 72 hour MTT assay, as previously described by Hansen, M. B., Nielsen,S. E., and Berg, K. ((1989) J. Immunol. Methods 119, 203-210) andincorporated herein by reference in its entirety, was used todifferentiate between a specific versus non-specific effect on cellkilling by Smac mimetics. Briefly, SK-OV-3 cells were seeded in 96-wellplates in McCoy's medium containing 10% fetal bovine serum albumin(20,000 per well) and incubated overnight at 37 C. The next day, testcompounds were added at various concentrations (0.003-10 μM) and theplates were incubated at 37° C. for an additional 72 hrs. 50 microlitersof 5 mg/mL MTT reagent was then added to each well and the plates wereincubated at 37° C. for 3 hours. At the end of the incubation period, 50microliters of DMSO was added to each well to dissolve cells and theoptical density (OD) of the wells was measured using a microplate reader(Victor² 1420, Wallac, Finland) at 535 nm. Cell survival (CS) wascalculated by the following equationCS=(OD treated well/mean OD control wells)×100%

Smac mimetic Entry 116 was tested using an ovarian cancer cell line,SK-OV-3, and MRC-5 cells were used as a normal cell control. FIG. 3shows that Entry 116 is 100,000× more effective at killing tumor cellsthen negative controls while normal (non-tumorigenic) cells remainunaffected.

The EC₅₀, defined as the drug concentration that results in 50% CS, wasderived by calculating the point where the dose-response curve crossesthe 50% CS point using GraphPad Prism. These results suggest that Smacmimetics that bind to XIAP can be used in the treatment of cancer eitheras monotherapy or in combination with chemotherapeutics.

Annexin V/Propidium Iodide Staining

To show the ability of Smac mimetics to induce apoptosis, AnnexinV-fluorescein isothiocyanate staining was performed. Briefly as permanufacturer's protocol (Invitrogen, Carlsbad, Calif.), cells wereexposed to various concentrations of Smac mimetics for 18-24 hrs. andthen removed from the assay plate by trypsinization. Cells were thenpelleted and resuspended in assay buffer (supplied by manufacturer).Annexin V and propidium iodide were added to the cell preparations andincubated for 1 hour in the dark at room temperature. Following theincubation additional buffer (200 μl) was then added to each tube, andthe samples were analyzed immediately by flow cytometry. In the presenceof Smac mimetics apoptosis was strongly promoted, as assessed byannexin/PI staining and analyzed by flow cytometry. The amplification inthe number of apoptotic cells (Annexin V positive/propidium iodidenegative—lower right quadrant) by IAP antagonists as compared to controlwas dose dependent and due to the induction of apoptosis and not viaincreasing the proportion of necrotic cells.

The chemopotentiating effect Smac mimetic using melanoma cells that havebeen shown to be resistant to the apoptotic effects of TRAIL, achemotherapeutic drug (Chawla-Sarkar. Clin. Cancer Res. (2001). Assaysfor cell proliferation (MTT assay, FIG. 4) revealed that when MDA-MB-231cells, a breast cancer cell line, were treated with a Smacpeptidomimetic of the invention, Entry 1, alone the cells were resistantto the antiproliferative effects of the Smac mimetic of the invention.In contrast, when Entry 1 was used in combination with TRAIL there was a1000 fold increase in the antiproliferative effect resulting in a100-fold increase in the cell killing as detected by the correspondingloss in colony formation. A control peptidomimetic (Entry 62) failed tosynergize with TRAIL and results (data not shown) indicate noanti-proliferative activity of Entry 62 alone or in combination withTRAIL. TRAIL alone induces little, if any, apoptosis of MDA MB-231 cellsafter 4 hours. Treatment with Entry 121 alone also failed to inducesignificant apoptosis (approximately 10% of cell total). In contrast, acombination of TRAIL with Entry 121 resulted in a 4 fold increase inapoptotic activity after 4 hours

The ability of cells to form viable colonies was analyzed by addingvarious concentrations of the compound in the presence and absence of0.4 ng/ml of TRAIL. Briefly, cells are seeded at 100 cells per well in a12 well format in 2 ml of growth medium. The medium is removed aftertwenty-four hours and replaced with Smac mimetics at variousconcentrations in growth medium with 1% DMSO. After 72 hours on test,the concentrations are removed and replaced with 2 ml. of growth medium.The plates are returned to the incubator for 7-10 days at which time thecolonies have multiplied to at least 10 cells per colony which can becounted by eye. The plates are stained with a 0.01% crystal violetsolution (wt:vol. in H₂O) for thirty minutes. The plates are washed withtap water to remove any residual crystal violet, dried and the coloniesare counted. Inhibition data were analyzed using the sigmoidaldose-response (variable slope) equation in GraphPad Prism (GraphPadSoftware, San Diego, Calif.). The 50% inhibitory concentration (EC₅₀)was the drug concentration that decreased the enzyme activity by 50%relative to control samples without drug.

Synergy was observed with topotecan and camptothecin, two examples oftopoisomerases inhibitors in a cytotoxicity study in T98G cells. Thehighest amount of synergy is 50-60% more cell death than would beexpected by adding together the cytotoxicity of each compound alone.Results show that both topotecan and camptothecin can actsynergistically with a Smac mimetic of the invention, such as Entry 1,for an enhancement of apoptosis. The total synergistic volume was 457,with the greatest synergism being about 30%-40% more cell death thanwould be expected by adding together the individual Smac and topotecancytotoxicities, between Entry 1 and a topoismerase inhibitor, such astopotecan.

To further assess potential drug-drug interactions a matrix of thepermutations of two-fold serial dilutions of each drug and a Snacmimetic were tested as well as the activity of each compound alone usinga program called MacSynergy II (Prichard, M. N., K. R. Aseltine, and C.Shipman, Jr. 1993. MacSynergy II. User's manual. Univ. of Michigan, AnnArbor). The synergy with paclitaxel and the Smac peptidomimetic Entry122 in OVCAR3 cells was tested. The highest amount of synergy detectedis 10-20% which indicates greater cell death than would be expected byadding together the cytotoxicity of either compound alone.

Taxanes are compounds that inhibit mitotsis by hinderingdepolymerization of the microtubule based spindle. These data weregenerated by testing various concentrations of a common taxane,paclitaxel, and a Smac mimetic. Paclitaxel the dosage ranged from about0.0 to about 500.0 nM. For Entry 122, the dosage range was about 125.0to about 8000.0 nM. The total synergistic volume was about 170.

The mechanism of action of platinum containing compounds is believed tobe by binding to DNA and interfering with its repair mechanism,eventually leading to cell death. The synergy with cisplatin and theSmac peptidomimetics in OVCAR-3 cells was tested The highest amount ofsynergy is 40-50% more cell death than would be expected by addingtogether the cytotoxicity of each compound alone. These data weregenerated by testing various concentrations of cisplatin and Smacmimetics drugs. For cisplatin the dosage range was about 0.0 to about166,500.0 nM. For Entry 122, the dosage range was about 500.0 to about32,000.0 nM. The total synergistic volume was about 434. Similar testswere performed with the combination of carboplatin and Smac mimetics.Synergy between a Smac peptidomimetic, Entry 122, and carboplatin.

This potent synergy makes possible the use of the Smac peptidomimetics,which are IAP antagonists, to improve the efficacy of the marketedanti-tumor compounds (such as but not limited to paclitaxel, cisplatinand carboplatin). This effected will allow the lowering the requireddose of the poorly tolerated anti-tumor compounds and/or by improvingthe responses rate at the marketed doses.

The present invention is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the appended claims.

What is claimed is:
 1. A compound of formula (II):

wherein R1 and R2 are independently H, tert-butoxycarbonyl, benzyloxycarbonyl, acetyl, trifluoroacetyl, alkyl, optionally-substituted alkyl, or

where R5a and R5b are independently H, alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl; or each optionally-substituted with hydroxyl, mercapto, halogen, amino, carboxyl, alkyl, haloalkyl, alkoxy, or alkylthio; or, optionally, R5a and R5b are connected by an alkylene, alkenylene, alkynylene bridge of 2 to 12 carbon atoms or an optionally-substituted alkylene, alkenylene, alkynylene bridge of 2 to 12 carbon atoms where one or more carbon atoms can be replaced with N, O, or S; R6a and R6b are independently H, tert-butoxycarbonyl, benzyloxycarbonyl, acetyl, trifluoroacetyl, alkyl, lower alkyl, optionally-substituted alkyl, or

where R7a and R7b are independently H, alkyl, cycloalkyl, haloalkyl; or R8a and R7a and R8b and R7b can independently or together form a ring; R8a and R8b are independently H, hydroxyl, alkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein each alkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, and heteroarylalkyl is optionally-substituted with halogen, hydroxyl, mercapto, carboxyl, alkyl, alkoxy, amino, and nitro; or R8a and R7a and R8b and R7b independently or together form a ring; R3a and R3b are independently H, halogen, alkyl, aryl, arylalkyl, amino, arylamino, arylalkylamino, hydroxy, alkyloxy, aryloxy, arylalkylhydroxy, dialkylamino, amido, sulfonamido, or amidino; m and n are independently 0, 1, 2, or 3; X and Y are independently O, N, S, or C═C; R9a, R9b, R10a, R10b are independently H, alkyl, optionally-substituted alkyl, aryl, heteroaryl, optionally-substituted aryl, heteroaryl, or R9a and R10a, independently or in parallel with R9b and R10b, can be linked by 4 to 8 optionally-substituted atoms such as C, N, O, or S, to form an aromatic or non-aromatic ring; and wherein when Wa and Wb are covalently bound, Wa and Wb together are a bond, alkylene, alkenylene, alkynylene, aryl, arylalkylene, arylalkylalkylene, heteroaryl, heteroarylalkylene, or an optionally-substituted alkylene, alkenylene, alkynylene chain of 2 to 12 carbon atoms where one or more carbon atoms can be replaced with N, O, or S; and R11a and R11b are independently absent, H, alkyl, optionally-substituted alkyl, hydroxyalkyl, alkoxyalkyl; or R11a and R11b together form an alkylene, alkenylene, alkynylene, or alkyloxyalkylene chain of 2 to 12 carbon atoms or an optionally-substituted alkylene, alkenylene, alkynylene, or alkyloxyalkylene chain of 2 to 12 carbon atoms where one or more carbon atoms can be replaced with N, O, or S; and when Wa and Wb are not covalently bound, Wa and Wb are independently H, Cl, Br, F, alkyl, CN, CO2H; and R11a and R11b together form an alkylene, alkenylene, alkynylene, or alkyloxyalkylene chain of 2 to 12 carbon atoms where one or more carbon atoms can be replaced with N, O, or S; or Wa can be H, Cl, Br, F, alkyl, CN, CO2H and Wb and R11a together are a bond, alkylene, alkenylene, alkynylene, aryl, arylalkylene, arylalkylalkylene, heteroaryl, heteroarylalkylene, or an optionally-substituted alkylene, alkenylene, alkynylene chain of 2 to 12 carbon atoms where one or more carbon atoms are replaced with N, O, or S; and R11b is absent or H, alkyl, optionally-substituted alkyl, hydroxyalkyl, alkoxyalkyl.
 2. The compound of claim 1, comprising a homodimer.
 3. The compound of claim 1, wherein R5a and R5b independently are selected from methyl, ethyl, isopropyl, isobutyl, sec-butyl, tert-butyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl.
 4. The compound of claim 1, wherein R7a and R7b is independently selected from methyl, fluoromethyl, difluoromethyl, ethyl, fluoroethyl, and cycloalkyl.
 5. The compound of claim 1, wherein the optionally substituted alkyl of R5a and R5b is independently selected from alkoxylated and hydroxylated alkyls.
 6. The compound of claim 1, wherein R3a and R3b are independently selected from H, hydroxy, alkyloxy, aryloxy, alkylamino, dialkylamino, amido, sulfonamido, or amidino.
 7. The compound of claim 1, wherein when Wa and Wb are covalently bound, Wa and Wb together are a bond, alkylene, alkenylene, alkynylene, aryl, arylalkylene, arylalkylalkylene, heteroaryl, heteroarylalkylene, or an optionally-substituted alkylene, alkenylene, alkynylene chain of 2 to 12 carbon atoms where one or more carbon atoms can be replaced with N, O, or S; and R11a and R11b are absent or independently H, lower alkyl, optionally-substituted alkyl, hydroxyalkyl, alkoxyalkyl; or R11a and R11b together form an alkylene, alkenylene, alkynylene, or alkyloxyalkyl chain of 2 to 12 carbon atoms where one or more carbon atoms are replaced with N, O, or S; and X and Y are selected from N, O, S, or C═C.
 8. The compound of claim 1, wherein when Wa and Wb are non-covalently bound, Wa and Wb are independently H, Cl, Br, F, alkyl, CN, CO2H; and R11a and R11b together form an alkylene, alkenylene, alkynylene, or alkyloxyalkyl chain of 2 to 12 carbon atoms where one or more carbon atoms are replaced with N, O, or S; and X and Y are selected from N, O, S, or C═C.
 9. The compound of claim 1, wherein when Wa is H, Cl, Br, F, alkyl, CN, CO2H and Wb and R11a together are a bond, alkylene, alkenylene, alkynylene, aryl, arylalkylene, arylalkylalkylene, heteroaryl, heteroarylalkylene, or an optionally-substituted alkylene, alkenylene, alkynylene chain of 2 to 12 carbon atoms where one or more carbon atoms are replaced with N, O, or S; and R11b is absent or H, alkyl, optionally-substituted alkyl, hydroxyalkyl, alkoxyalkyl and X and Y are selected from N, O, S, or C═C.
 10. The compound of claim 1, of the formula (III):

wherein R1 and R2 are, independently, H, tert-butoxycarbonyl, benzyloxycarbonyl, acetyl, trifluoroacetyl, alkyl, optionally-substituted alkyl, or:

where R5a and R5b are independently H, alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl; or each optionally-substituted with hydroxyl, mercapto, halogen, amino, carboxyl, alkyl, haloalkyl, alkoxy, or alkylthio; or, optionally, R5a and R5b are connected by an alkylene, alkenylene, alkynylene bridge of 2 to 12 carbon atoms or optionally-substituted alkylene, alkenylene, alkynylene bridge of 2 to 12 carbon atoms where one or more carbon atoms can be replaced with N, O, or S; R6a and R6b are, independently, H, tert-butoxycarbonyl, benzyloxycarbonyl, acetyl, trifluoroacetyl, alkyl, lower alkyl, optionally-substituted alkyl, or:

where R7a and R7b are independently H, alkyl, cycloalkyl, haloalkyl; or R8a and R7a and R8b and R7b can independently or together form a ring; R8a and R8b are independently H, hydroxyl, alkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein each alkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, and heteroarylalkyl is optionally-substituted with halogen, hydroxyl, mercapto, carboxyl, alkyl, alkoxy, amino, and nitro; or R8a and R7a and R8b and R7b can independently or together form a ring; R3a and R3b are independently H, halogen, alkyl, aryl, arylalkyl, amino, arylamino, arylalkylamino, hydroxy, alkyloxy, aryloxy, arylalkylhydroxy, dialkylamino, amido, sulfonamido, or amidino; m and n are independently 0, 1, 2, or 3; X and Y are independently O, N, S, or C═C; R12a, R12b, R13a, R13b, R14a, R14b are, independently, H, Cl, Br, F, alkyl, cycloalkyl, hydroxyl, alkoxy, amino, alkylamino, cyano, or CO₂H; and wherein when Wa and Wb are covalently bound, Wa and Wb together are a bond, alkylene, alkenylene, alkynylene, aryl, arylalkylene, arylalkylalkylene, heteroaryl, heteroarylalkylene, or an optionally-substituted alkylene, alkenylene, alkynylene chain of 2 to 12 carbon atoms where one or more carbon atoms can be replaced with N, O, or S; and R11a and R11b are independently absent or H, alkyl, optionally-substituted alkyl, hydroxyalkyl, alkoxyalkyl; or R11a and R11b together form an alkylene, alkenylene, alkynylene, or alkyloxyalkylene chain of 2 to 12 carbon atoms or an optionally-substituted alkylene, alkenylene, alkynylene, or alkyloxyalkylene chain of 2 to 12 carbon atoms where one or more carbon atoms can be replaced with N, O, or S; and when Wa and Wb are not covalently bound, Wa and Wb are independently H, Cl, Br, F, alkyl, CN, CO2H; and R11a and R11b together form an alkylene, alkenylene, alkynylene, or alkyloxyalkylene chain of 2 to 12 carbon atoms or optionally substituted alkylene, alkenylene, alkynylene, or alkyloxyalkylene chain of 2 to 12 carbon atoms where one or more carbon atoms are replaced with N, O, or S; or Wa is H, Cl, Br, F, alkyl, CN, CO₂H and Wb and R11a together are a bond, alkylene, alkenylene, alkynylene, aryl, arylalkylene, arylalkylalkylene, heteroaryl, heteroarylalkylene, or an optionally-substituted alkylene, alkenylene, alkynylene chain of 2 to 12 carbon atoms where one or more carbon atoms can be replaced with N, O, or S; and R11b is absent or H, alkyl, optionally-substituted alkyl, hydroxyalkyl, alkoxyalkyl.
 11. The compound of claim 10, wherein R3a and R3b are independently selected from H, hydroxy, alkyloxy, aryloxy, alkylamino, dialkylamino, amido, sulfonamido, or amidino.
 12. The compound of claim 11, wherein the optionally substituted alkyl of R5a and R5b independently is selected from alkoxylated and hydroxylated alkyls.
 13. A compound of claim 1, of formula (IV):

where R5a and R5b are independently H, alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl; or each optionally-substituted with hydroxyl, mercapto, halogen, amino, carboxyl, alkyl, haloalkyl, alkoxy, or alkylthio; or, optionally, R5a and R5b are connected by an alkylene, alkenylene, alkynylene of 2 to 12 carbon atoms or optionally-substituted alkylene, alkenylene, alkynylene bridge of 2 to 12 carbon atoms where one or more carbon atoms can be replaced with N, O, or S; where R7a and R7b are independently H, alkyl, cycloalkyl, haloalkyl; or R8a and R7a and R8b and R7b can independently or together form a ring; R8a and R8b are independently H, hydroxyl, alkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein each alkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, and heteroarylalkyl is optionally-substituted with halogen, hydroxyl, mercapto, carboxyl, alkyl, alkoxy, amino, and nitro; or R8a and R7a and R8b and R7b can independently or together form a ring; R3a and R3b are independently H, halogen, alkyl, aryl, arylalkyl, amino, arylamino, arylalkylamino, hydroxy, alkyloxy, aryloxy, arylalkylhydroxy, dialkylamino, amido, sulfonamido, or amidino; X and Y are independently O, N, S, or C═C; m and n are independently 0, 1, 2, or 3; and R12a, R12b, R13a, R13b, R14a, R14b are independently H, Cl, Br, F, alkyl, cycloalkyl, hydroxyl, alkoxy, amino, alkylamino, cyano, or CO₂H; and wherein when Wa and Wb are covalently bound, Wa and Wb are a bond, alkylene, alkenylene, alkynylene, aryl, arylalkylene, arylalkylalkylene, heteroaryl, heteroarylalkylene, or an optionally-substituted alkylene, alkenylene, alkynylene chain of 2 to 12 carbon atoms where one or more carbon atoms are replaced with N, O, or S; and R11a and R11b are independently absent, H, alkyl, optionally-substituted alkyl, hydroxyalkyl, alkoxyalkyl; or R11a and R11b together form an alkylene, alkenylene, alkynylene, or alkyloxyalkylene chain of 2 to 12 carbon atoms where one or more carbon atoms are replaced with N, O, or S; and when Wa and Wb are not covalently bound, Wa and Wb are independently H, Cl, Br, F, alkyl, CN, CO2H; and R11a and R11b together form an alkylene, alkenylene, alkynylene, or alkyloxyalkylene chain of 2 to 12 carbon atoms or optionally substituted alkylene, alkenylene, alkynylene, or alkyloxyalkylene chain of 2 to 12 carbon atoms where one or more carbon atoms are replaced with N, O, or S; or Wa is H, Cl, Br, F, alkyl, CN, CO₂H and Wb and R11a together are a bond, alkylene, alkenylene, alkynylene, aryl, arylalkylene, arylalkylalkylene, heteroaryl, heteroarylalkylene of 2 to 12 carbon atoms, or an optionally-substituted alkylene, alkenylene, alkynylene chain of 2 to 12 carbon atoms where one or more carbon atoms can be replaced with N, O, or S; and R11b is absent or H, alkyl, optionally-substituted alkyl, hydroxyalkyl, alkoxyalkyl.
 14. The compound of claim 13, wherein the optionally substituted alkyl of R5a and R5b independently is selected from alkoxylated and hydroxylated alkyls.
 15. The compound of claim 14, wherein R3a and R3b are selected from H, hydroxy, alkyloxy, aryloxy, alkylamino, dialkylamino, amido, sulfonamido, or amidino.
 16. The compound of claim 1, of formula (V):

where R5a and R5b are independently H, alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl; or each optionally-substituted with hydroxyl, mercapto, halogen, amino, carboxyl, alkyl, haloalkyl, alkoxy, or alkylthio; or, optionally, R5a and R5b are connected by an alkylene, alkenylene, alkynylene bridge of 2 to 12 carbon or optionally-substituted alkylene, alkenylene, alkynylene bridge of 2 to 12 carbon atoms where one or more carbon atoms can be replaced with N, O, or S; where R7a and R7b are independently H, alkyl, cycloalkyl, haloalkyl; or R8a and R7a and R8b and R7b can independently or together form a ring; R8a and R8b are independently H, hydroxyl, alkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein each alkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, and heteroarylalkyl is optionally-substituted with halogen, hydroxyl, mercapto, carboxyl, alkyl, alkoxy, amino, and nitro; or R8a and R7a and R8b and R7b can independently or together form a ring; R3a and R3b are independently H, halogen, alkyl, aryl, arylalkyl, amino, arylamino, arylalkylamino, hydroxy, alkyloxy, aryloxy, arylalkylhydroxy, dialkylamino, amido, sulfonamido, or amidino; m and n are independently 0, 1, 2, or 3; and R12a, R12b, R13a, R13b, R14a, R14b are independently H, Cl, Br, F, alkyl, cycloalkyl, hydroxyl, alkoxy, amino, alkylamino, cyano, or CO₂H; and wherein when Wa and Wb are covalently bound, Wa and Wb together are a bond, alkylene, alkenylene, alkynylene, aryl, arylalkylene, arylalkylalkylene, heteroaryl, heteroarylalkylene, or an optionally-substituted alkylene, alkenylene, alkynylene chain of 2 to 12 carbon atoms where one or more carbon atoms can be replaced with N, O, or S; and R11a and R11b are absent or independently H, alkyl, optionally-substituted alkyl, hydroxyalkyl, alkoxyalkyl; or R11a and R11b together form an alkylene, alkenylene, alkynylene, or alkyloxyalkylene chain of 2 to 12 or optionally-substituted alkylene, alkenylene, alkynylene, or alkyloxyalkylene chain of 2 to 12 carbon atoms where one or more carbon atoms is replaced with N, O, or S; and when Wa and Wb are not covalently bound, Wa and Wb are independently H, Cl, Br, F, alkyl, CN, CO2H; and R11a and R11b together form an alkylene, alkenylene, alkynylene, or alkyloxyalkylene chain of 2 to 12 carbon atoms or optionally substituted alkylene, alkenylene, alkynylene, or alkyloxyalkylene chain of 2 to 12 carbon atoms where one or more carbon atoms is replaced with N, O, or S; or Wa is H, Cl, Br, F, alkyl, CN, CO2H and Wb and R11a together are a bond, alkylene, alkenylene, alkynylene, aryl, arylalkylene, arylalkylalkylene, heteroaryl, heteroarylalkylene, or an optionally-substituted alkylene, alkenylene, alkynylene chain of 2 to 12 carbon atoms where one or more carbon atoms are replaced with N, O, or S; and R11b is absent or H, alkyl, optionally-substituted alkyl, hydroxyalkyl, alkoxyalkyl.
 17. The compound of claim 16, wherein the optionally substituted alkyl of Rya and R5b independently is selected from alkoxylated and hydroxylated alkyls.
 18. The compound of claim 16, wherein R3a and R3b are selected from H, hydroxy, alkyloxy, aryloxy, alkylamino, dialkylamino, amido, sulfonamido, or amidino.
 19. The compound of claim 1, of formula (VI):

where R5a and R5b are independently H, alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl; or each optionally-substituted with hydroxyl, mercapto, halogen, amino, carboxyl, alkyl, haloalkyl, alkoxy, or alkylthio; or, optionally, R5a and R5b are connected by an alkylene, alkenylene, alkynylene bridge of 2 to 12 carbon atoms or optionally-substituted alkylene, alkenylene, alkynylene bridge of 2 to 12 carbon atoms where one or more carbon atoms can be replaced with N, O, or S; where R7a and R7b are independently H, alkyl, cycloalkyl, haloalkyl; or R8a and R7a and R8b and R7b can independently or together form a ring; R8a and R8b are independently H, hydroxyl, alkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein each alkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, and heteroarylalkyl is optionally-substituted with halogen, hydroxyl, mercapto, carboxyl, alkyl, alkoxy, amino, and nitro; or R8a and R7a and R8b and R7b can independently or together form a ring; R3a and R3b are independently H, halogen, alkyl, aryl, arylalkyl, amino, arylamino, arylalkylamino, hydroxy, alkyloxy, aryloxy, arylalkylhydroxy, dialkylamino, amido, sulfonamido, or amidino; X is O, N, S, or C═C; and R12a, R12b, R13a, R13b, R14a, R14b are independently H, Cl, Br, F, alkyl, cycloalkyl, hydroxyl, alkoxy, amino, alkylamino, cyano, or CO₂H; and wherein Wa and Wb are not covalently bound, Wa and Wb are independently H, Cl, Br, F, alkyl, CN, CO₂H; and Wb and R11a together are a bond, alkylene, alkenylene, alkynylene, aryl, arylalkylene, arylalkylalkylene, heteroaryl, heteroarylalkylene, or an optionally-substituted alkylene, alkenylene, alkynylene chain of 2 to 12 carbon atoms where one or more carbon atoms is replaced with N, O, or S; and R11b is absent or H, alkyl, optionally-substituted alkyl, hydroxyalkyl, alkoxyalkyl.
 20. A pharmaceutical composition comprising: a compound selected from the group consisting of compounds of formula

and a pharmaceutically acceptable excipient, wherein R1 and R2 are independently H, tert-butoxycarbonyl, benzyloxycarbonyl, acetyl, trifluoroacetyl, alkyl, optionally-substituted alkyl, or

where R5a and R5b are independently H, alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl; or each optionally-substituted with hydroxyl, mercapto, halogen, amino, carboxyl, alkyl, haloalkyl, alkoxy, or alkylthio; or, optionally, R5a and R5b are connected by an alkylene, alkenylene, alkynylene bridge of 2 to 12 carbon atoms or an optionally-substituted alkylene, alkenylene, alkynylene bridge of 2 to 12 carbon atoms where one or more carbon atoms can be replaced with N, O, or S; R6a and R6b are independently H, tert-butoxycarbonyl, benzyloxycarbonyl, acetyl, trifluoroacetyl, alkyl, lower alkyl, optionally-substituted alkyl, or

where R7a and R7b are independently H, alkyl, cycloalkyl, haloalkyl; or R8a and R7a and R8b and R7b can independently or together form a ring; R8a and R8b are independently H, hydroxyl, alkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein each alkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, and heteroarylalkyl is optionally-substituted with halogen, hydroxyl, mercapto, carboxyl, alkyl, alkoxy, amino, and nitro; or R8a and R7a and R8b and R7b independently or together form a ring; R3a and R3b are independently H, halogen, alkyl, aryl, arylalkyl, amino, arylamino, arylalkylamino, hydroxy, alkyloxy, aryloxy, arylalkylhydroxy, dialkylamino, amido, sulfonamido, or amidino; m and n are independently 0, 1, 2, or 3; X and Y are independently O, N, S, or C═C; R9a, R9b, R10a, R10b are independently H, alkyl, optionally-substituted alkyl, aryl, heteroaryl, optionally-substituted aryl, heteroaryl, or R9a and R10a, independently or in parallel with R9b and R10b, can be linked by 4 to 8 optionally-substituted atoms such as C, N, O, or S, to form an aromatic or non-aromatic ring; R12a, R12b, R13a, R13b, R14a, R14b are, independently, H, Cl, Br, F, alkyl, cycloalkyl, hydroxyl, alkoxy, amino, alkylamino, cyano, or CO₂H; and wherein when Wa and Wb are covalently bound, Wa and Wb together are a bond, alkylene, alkenylene, alkynylene, aryl, arylalkylene, arylalkylalkylene, heteroaryl, heteroarylalkylene, or an optionally-substituted alkylene, alkenylene, alkynylene chain of 2 to 12 carbon atoms where one or more carbon atoms can be replaced with N, O, or S; and R11a and R11b are independently absent, H, alkyl, optionally-substituted alkyl, hydroxyalkyl, alkoxyalkyl; or R11a and R11b together form an alkylene, alkenylene, alkynylene, or alkyloxyalkylene chain of 2 to 12 carbon atoms or an optionally-substituted alkylene, alkenylene, alkynylene, or alkyloxyalkylene chain of 2 to 12 carbon atoms where one or more carbon atoms can be replaced with N, O, or S; and when Wa and Wb are not covalently bound, Wa and Wb are independently H, Cl, Br, F, alkyl, CN, CO2H; and R11a and R11b together form an alkylene, alkenylene, alkynylene, or alkyloxyalkylene chain of 2 to 12 carbon atoms where one or more carbon atoms can be replaced with N, O, or S; or Wa can be H, Cl, Br, F, alkyl, CN, CO2H and Wb and R11a together are a bond, alkylene, alkenylene, alkynylene, aryl, arylalkylene, arylalkylalkylene, heteroaryl, heteroarylalkylene, or an optionally-substituted alkylene, alkenylene, alkynylene chain of 2 to 12 carbon atoms where one or more carbon atoms are replaced with N, O, or S; and R11b is absent or H, alkyl, optionally-substituted alkyl, hydroxyalkyl, alkoxyalkyl.
 21. A method for inducing apoptosis in a cell comprising contacting the cell with a compound selected from the group consisting of compounds of formula

wherein R1 and R2 are independently H, tert-butoxycarbonyl, benzyloxycarbonyl, acetyl, trifluoroacetyl, alkyl, optionally-substituted alkyl, or

where R5a and R5b are independently H, alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl; or each optionally-substituted with hydroxyl, mercapto, halogen, amino, carboxyl, alkyl, haloalkyl, alkoxy, or alkylthio; or, optionally, R5a and R5b are connected by an alkylene, alkenylene, alkynylene bridge of 2 to 12 carbon atoms or an optionally-substituted alkylene, alkenylene, alkynylene bridge of 2 to 12 carbon atoms where one or more carbon atoms can be replaced with N, O, or S; R6a and R6b are independently H, tert-butoxycarbonyl, benzyloxycarbonyl, acetyl, trifluoroacetyl, alkyl, lower alkyl, optionally-substituted alkyl, or

where R7a and R7b are independently H, alkyl, cycloalkyl, haloalkyl; or R8a and R7a and R8b and R7b can independently or together form a ring; R8a and R8b are independently H, hydroxyl, alkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein each alkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, and heteroarylalkyl is optionally-substituted with halogen, hydroxyl, mercapto, carboxyl, alkyl, alkoxy, amino, and nitro; or R8a and R7a and R8b and R7b independently or together form a ring; R3a and R3b are independently H, halogen, alkyl, aryl, arylalkyl, amino, arylamino, arylalkylamino, hydroxy, alkyloxy, aryloxy, arylalkylhydroxy, dialkylamino, amido, sulfonamido, or amidino; m and n are independently 0, 1, 2, or 3; X and Y are independently O, N, S, or C═C; R9a, R9b, R10a, R10b are independently H, alkyl, optionally-substituted alkyl, aryl, heteroaryl, optionally-substituted aryl, heteroaryl, or R9a and R10a, independently or in parallel with R9b and R10b, can be linked by 4 to 8 optionally-substituted atoms such as C, N, O, or S, to form an aromatic or non-aromatic ring; R12a, R12b, R13a, R13b, R14a, R14b are, independently, H, Cl, Br, F, alkyl, cycloalkyl, hydroxyl, alkoxy, amino, alkylamino, cyano, or CO₂H; and wherein when Wa and Wb are covalently bound, Wa and Wb together are a bond, alkylene, alkenylene, alkynylene, aryl, arylalkylene, arylalkylalkylene, heteroaryl, heteroarylalkylene, or an optionally-substituted alkylene, alkenylene, alkynylene chain of 2 to 12 carbon atoms where one or more carbon atoms can be replaced with N, O, or S; and R11a and R11b are independently absent, H, alkyl, optionally-substituted alkyl, hydroxyalkyl, alkoxyalkyl; or R11a and R11b together form an alkylene, alkenylene, alkynylene, or alkyloxyalkylene chain of 2 to 12 carbon atoms or an optionally-substituted alkylene, alkenylene, alkynylene, or alkyloxyalkylene chain of 2 to 12 carbon atoms where one or more carbon atoms can be replaced with N, O, or S; and when Wa and Wb are not covalently bound, Wa and Wb are independently H, Cl, Br, F, alkyl, CN, CO2H; and R11a and R11b together form an alkylene, alkenylene, alkynylene, or alkyloxyalkylene chain of 2 to 12 carbon atoms where one or more carbon atoms can be replaced with N, O, or S; or Wa can be H, Cl, Br, F, alkyl, CN, CO2H and Wb and R11a together are a bond, alkylene, alkenylene, alkynylene, aryl, arylalkylene, arylalkylalkylene, heteroaryl, heteroarylalkylene, or an optionally-substituted alkylene, alkenylene, alkynylene chain of 2 to 12 carbon atoms where one or more carbon atoms are replaced with N, O, or S; and R11b is absent or H, alkyl, optionally-substituted alkyl, hydroxyalkyl, alkoxyalkyl. 